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Impaired Phagosomal Maturation in
Neutrophils Leads to Periodontitis in
Lysosomal-Associated Membrane Protein-2
Knockout Mice
Wouter Beertsen, Marion Willenborg, Vincent Everts,
Angelika Zirogianni, Rainer Podschun, Bernd Schröder,
Eeva-Liisa Eskelinen and Paul Saftig
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2008 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2008; 180:475-482; ;
doi: 10.4049/jimmunol.180.1.475
http://www.jimmunol.org/content/180/1/475
The Journal of Immunology
Impaired Phagosomal Maturation in Neutrophils Leads to
Periodontitis in Lysosomal-Associated Membrane Protein-2
Knockout Mice1
Wouter Beertsen,2§ Marion Willenborg,2* Vincent Everts,¶ Angelika Zirogianni,§
Rainer Podschun,† Bernd Schröder,* Eeva-Liisa Eskelinen,‡ and Paul Saftig3*
P
eriodontitis is an infectious disease that is one of the most
widespread diseases worldwide (1). It is estimated to affect
up to 15% of the adult dentate population (2). Periodontitis is
an inflammatory disease of the supporting tissues of the teeth leading
to resorption of alveolar bone and eventually tooth loss. The disease
is characterized by a constant interaction between pathogenic bacteria
and the host defense mechanisms. In health, host immune responses
are sufficient to hold in check the pathogenic potential of both the
normal resident microbial flora and exogenous microbial pathogens.
Complex inflammatory and immune reactions are involved in the progression of periodontitis. Polymorphonuclear leukocytes (PMNs)4
and circulating neutrophils constitute the first defense barrier against
the oral bacterial challenge in the periodontium (3). They are rapidly
*Biochemical Institute, Christian-Albrechts-University Kiel and †Institute of Infection Medicine, University Hospital Schleswig-Holstein, Kiel, Germany; ‡Department
of Biological and Environmental Sciences, Division of Biochemistry, University of
Helsinki, Helsinki, Finland; and §Department of Periodontology and ¶Department of
Oral Cell Biology, Academic Centre for Dentistry Amsterdam, Universiteit van Amsterdam and Vrije Universiteit, Amsterdam, The Netherlands
Received for publication October 17, 2007. Accepted for publication October
22, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the Deutsche Forschungsgemeinschaft DFG
SA683/6 –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: [email protected]
4
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
www.jimmunol.org
recruited from the blood to the site at risk, and then phagocytose and
kill the intruders. Neutrophils may release proinflammatory mediators
that amplify the local inflammatory reaction, further promoting
leukocyte and platelet recruitment. Quantitative or qualitative
abnormalities of the PMNs may, therefore, have an effect on the
accumulation of plaque in the supra and subgingival regions.
Malfunctioning of PMNs including a disturbed adhesion to the
endothelium, chemotaxis, detoxification of bacterial products,
phagocytosis, or degranulation have been associated with early
onset periodontitis (4 –7).
Phagosome-lysosome fusion bestows on the phagocytic vacuole
the lytic properties for efficient removal of internalized pathogens.
Lysosomes play a crucial role in the oxygen-independent killing of
bacteria, which is believed to be an important killing mechanism in
the oxygen-deprived periodontal pocket (8).
The limiting membrane of the lysosomal compartment is
thought to be of importance for phagosome maturation (9).
Lysosome-associated membrane protein (LAMP)-2 is a highly
glycosylated protein. It is an abundant and important constituent of the lysosomal membrane involved in lysosomal biogenesis 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 vacuoles
accumulate due to an impaired proteolysis of long-lived proteins in a
number of tissues, especially in myocytes, cardiomyocytes, and hepatocytes (10). In the latter cells, we observed an elevated secretion of
lysosomal enzymes, impaired processing of cathepsin D, and an abnormal retention of mannose-6-phosphate receptors in autophagic
vacuoles (13).
We report that LAMP-2-deficient mice show an increased susceptibility to periodontitis. PMNs isolated from these mice show
an impaired fusion between phagosomes and lysosomes leading to
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Inflammatory periodontal diseases constitute one of the most common infections in humans, resulting in the destruction of
the supporting structures of the dentition. Circulating neutrophils are an essential component of the human innate immune
system. We observed that mice deficient for the major lysosomal-associated membrane protein-2 (LAMP-2) developed severe
periodontitis early in life. This development was accompanied by a massive accumulation of bacterial plaque along the tooth
surfaces, gingival inflammation, alveolar bone resorption, loss of connective tissue fiber attachment, apical migration of
junctional epithelium, and pathological movement of the molars. The inflammatory lesions were dominated by polymorphonuclear leukocytes (PMNs) apparently being unable to efficiently clear bacterial pathogens. Systemic treatment of
LAMP-2-deficient mice with antibiotics prevented the periodontal pathology. Isolated PMNs from LAMP-2-deficient mice
showed an accumulation of autophagic vacuoles and a reduced bacterial killing capacity. Oxidative burst response was not
altered in these cells. Latex bead and bacterial feeding experiments showed a reduced ability of the phagosomes to acquire
an acidic pH and late endocytic markers, suggesting an impaired fusion of late endosomes-lysosomes with phagosomes. This
study underlines the importance of LAMP-2 for the maturation of phagosomes in PMNs. It also underscores the requirement
of lysosomal fusion events to provide sufficient antimicrobial activity in PMNs, which is needed to prevent periodontal
disease. The Journal of Immunology, 2008, 180: 475– 482.
476
a decreased bacterial killing capacity. This impairment is the likely
cause of the development of severe periodontitis.
Materials and Methods
Mice, cell lines, and Abs
Tissue processing, measurements, and statistical analysis
Mice were killed between 7 wk and 12 mo after birth. Upper and lower
jaws were fixed in 4% paraformaldehyde and 1% glutaraldehyde in 0.1 M
sodium cacodylate buffer (pH 7.4). Jaws were micrographed and visualized
by high resolution MicroCT. Following demineralization in EDTA, jaws
were postfixed in 1% OsO4 and embedded in epoxy resin. Sections were
cut parallel to the longitudinal axis of the molars and stained with methylene blue. One midsagittal section of each molar block (four per animal)
was subjected to image analysis using the Leica-Qwin-Pro software (objectives ⫻6.3 and ⫻10). To this end, the area between the first and second
molars, the standard interproximal area, was assessed for microbial plaque,
infiltrated interdental epithelium, supraalveolar connective tissue, alveolar
bone, and periodontal ligament. In addition, connective tissue attachment
and bone levels were determined distal to the first molar and mesial to the
second molar in each molar block (four per animal). The connective tissue
attachment level was defined as the distance from cemento-enamel junction
(CEJ) to the apical termination of the junctional epithelium. Bone level was
defined as the distance between CEJ and bone crest. Data were analyzed by
Student’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, supragingival
plaque samples were taken from the interdental areas (lingual aspect) of the
molars using paper points. The samples were cultured aerobically and
anaerobically according to routine laboratory procedures for detection of
periodontal pathogens.
Latex bead phagocytosis and phagocytosis with Aggregatibacter
(Actinobacillus) actinomycetemcomitans
Latex beads (3 ␮m; Sigma-Aldrich) were opsonized in RPMI 1640 medium containing 10% human serum for 30 min at 37°C and were washed
three times with medium. The 1–5 ⫻ 105 peritoneal PMNs per well were
cultured in a 24-well plate with RPMI 1640 medium (without antibiotics
and FCS) to adhere to the surface. Opsonized beads (0.05% solid) in serum-free RPMI 1640 were then added. Phagocytosis was synchronized by
spinning at 300 ⫻ g for 1 min. To induce internalization, cells were incubated 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 for
immunofluorescence. Before permeabilizing the cells external beads were
labeled with fluorophore-conjugated goat anti-human IgG (1:500) in PBS
for 1 h at room temperature.
A. actinomycetemcomitans bacteria were cocultured with peritoneal
neutrophils isolated from wild-type and LAMP-2-deficient mice (ratio
1:50) at 37°C and 5% CO2 for 2 h. Extracellular bacteria were eliminated
by washing with PBS and gentamicin treatment. After different time points
the cells were fixed with 4% paraformaldehyde in PBS and examined
microscopically.
Immunofluorescence
PMNs were cultured on coverslips for 2 h and fixed with 4% paraformaldehyde in PBS for 20 –30 min at room temperature. Cells were permeabilized 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 488
or 594 (Molecular Probes) were used. Nuclei were stained with DAPI
(4⬘,6-diamidino-2-phenylindole; Sigma-Aldrich). Acidic compartments
were labeled by incubating living cells in RPMI 1640 containing 20 mM
HEPES (pH 7.4), and 0.1 mM DAMP (N-(3-((2,4-DNP)aminopropyl)-N(3-aminopropyl)methylamine; Molecular Probes). The coverslips were
mounted with Mowiol (Calbiochem) containing the anti-fading reagent
DABCO (1,4 diazobicyclo-(2.2.2) octane; Sigma-Aldrich), and viewed
with an Axiovert 200M fluorescence microscope (Zeiss) with or without an
Apotome device for optical sectioning.
Electron microscopy
For electron microscopy, ultrathin sections were cut from the interdental
region between the first and second mandibular and maxillary molars. Sections were stained with uranyl acetate and lead citrate and examined in a
Philips EM 420. Isolated PMNs were fixed in 4% formaldehyde and 1%
glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) and processed
for LX-112 embedding. For autophagic vacuole quantification, ultrathin
sections of PMNs were scored under the microscope. The number of autophagic vacuole profiles was counted in at least 100 cell profiles per
phenotype.
Killing assays
A total of 1 ⫻ 108 Escherichia coli cells were opsonized in RPMI 1640
medium containing 10% human serum for 30 min at 37°C and were
washed three times with medium. Peritoneal PMNs (5 ⫻ 105 per well)
were cultured in a 24-well plate with RPMI 1640 medium (without antibiotics and FCS) to adhere to the surface before 5 ⫻ 106 E. coli were
added. Phagocytosis was synchronized by spinning at 720 ⫻ g for 2 min.
Cells were incubated at 37°C for 1 h. Phagocytosis was stopped by putting
the cells on ice. Extracellular bacteria were eliminated by washing with
PBS and incubation with 100 ␮g/ml gentamicin for 30 min. The PMNs
were now cultured in RPMI 1640 containing 0.1% FCS. After various time
periods the medium was removed, and 0.1% BSA in H2Odest was added to
lyse 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 by
incubation 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 agar
plates 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% yeast
extract (BD Biosciences) and 2.5% glucose (Merck) or on Columbia blood
agar plates (Oxoid) at 37°C in 5% CO2. A total of 1 ⫻ 106 peritoneal
neutrophils were incubated with opsonized A. actinomycetemcomitans (ratio 1:50) in PBS at 37°C and 5% CO2 for 120 or 240 min. Phagocytosis was
synchronized by spinning the mixture at 720 ⫻ g for 2 min. Serial dilutions
of 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 in
each sample was then determined by comparing the number of CFU from the
control sample without added neutrophils (100% viability) to the number of
CFU obtained for A. actinomycetemcomitans incubated with neutrophils.
Hydrogen peroxide production by PMNs
The assay was performed according to Pick and Mizel (16). Peritoneal
PMNs (1 ⫻ 105 per well) were cultured in a 96-well plate with RPMI 1640
medium (without antibiotics and FCS) for 2 h to allow adherence before
nonadherent cells were removed by shaking the plates and washing the
wells three times with 0.1-ml volumes of warm phenol red-free HBSS. A
total of 2 ⫻ 107 opsonized E. coli bacteria XL1 blue or 0.1 mg of zymosan
was added in 100 ␮l of phenol red solution (140 mM NaCl, 10 mM
NaH2PO4, 5.5 mM sucrose, 0.56 mM phenol rot (pH 7.0), sterile filtered
0.22 ␮m) containing 19 U/ml HRP. After 1 h at 37°C, the reaction was
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LAMP-2-deficient mice were generated as previously described (10). The
mice were maintained in a conventional facility. For antibiotic treatment
selected mice received amoxicillin in the drinking water before birth (the
pregnant mothers) and immediately after birth (in a concentration of 5 mg/L).
Mouse primary neutrophil granulocytes were isolated from 2- to 4-moold mice. Mice were injected i.p. with 1 ml of sterile 4% Brewer’s thioglycolate solution (Difco/BD Biosciences). After 4 h the mice were killed,
and cells were recovered by peritoneal lavage using 5–10 ml of ice-cold
PBS/0.02% EDTA (w/v). The number of viable cells was checked by staining the cells with 0.4% trypan blue (Invitrogen Life Technologies). The
neutrophil yield was determined by flow cytometry after staining with PEconjugated anti-Gr-1 Ab (Miltenyi Biotec) and with FITC-conjugated
F4/80 Ab (Serotec) after three washes in PBS/0.5% BSA/0.02% NaN3.
Before staining, nonspecific binding of Abs was blocked with the anti-Fc
receptor Ab 2.4G2 (BD Pharmingen). Flow cytometric measurements were
performed using FACScan (BD Biosciences). Unless stated otherwise, the
cells were cultured in RPMI 1640 containing 10% FCS and penicillin/
streptomycin (Invitrogen Life Technologies) for 2 h to adhere to the surface. Before their use for the experiments nonadherent cells were washed
away, which resulted in ⬎95% pure PMN cultures.
The following Abs were used in this study: rabbit antiserum against
mouse cathepsin D (15); rat anti-mouse LAMP-1 and rat anti-mouse
LAMP-2 (Developmental Studies Hybridoma Bank); rabbit anti-LC3 from
I. 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 (ICN
Biomedicals). Alexa Fluor-conjugated secondary Abs were from Molecular Probes (Invitrogen Life Technologies).
LAMP-2 DEFICIENCY AND PERIODONTAL DISEASE
The Journal of Immunology
stopped by adding 10 ␮l of 1 M NaOH per well. The absorbance was
measured in a microtiter plate reader at 600 nm.
Results
LAMP-2-deficient mice display periodontitis
and they showed fenestrations. PMNs (without showing signs of
bacterial phagocytosis) were abundantly present in contact with
the blood vessel wall, within the collagenous fiber framework of
the gingiva, and within the epithelium. The area occupied by (infiltrated) epithelium was significantly larger for LAMP-2 knockout
mice compared with wild-type mice ( p ⬍ 0.001) (Fig. 1K). Supraalveolar connective tissue was significantly more extensive in
knockout mice compared with wild-type animals ( p ⬍ 0.05) (Fig.
1F). The surface area of alveolar bone in the interproximal area of
the wild-type animals was larger compared with that of the knockout mice ( p ⬍ 0.001) (Fig. 1K). The surface area of periodontal
ligament gave higher values for the knockout than the wild-type
mice ( p ⬍ 0.05) (Fig. 1K).
Analysis of histomorphometric parameters showed considerable
loss of connective tissue attachment (the distance between the CEJ to
the apical termination of junctional epithelium) in knockout animals
(Fig. 1L). Also the alveolar bone crest was displaced in the apical
direction (Fig. 1L), whereas the total molar root length (CEJ-apex)
was about the same (Fig. 1L). Plaque growth and extent of connective
tissue attachment loss did not show statistically significant differences
between 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 to
phagocytose pathogens but unable to efficiently kill the microorganisms. To investigate this, we isolated PMNs from wild-type
and this LAMP-2-deficient mice. After attachment of these cells
they were incubated for 1 h with E. coli cells. After 1 h phagocytosis, extracellular bacteria were killed by incubation for 30 min
with gentamicin. Cells were lysed and plated on bacterial plates to
estimate CFUs after 0, 30, and 60 min of subsequent incubation
(Fig. 2A). Wild-type PMNs were able to phagocytose and kill the
ingested bacteria as expected. LAMP-2-deficient PMNs were significantly less effective in killing bacteria. Even 60 min after incubation, the number of viable bacteria was comparable to the
number of bacteria at 0 min of wild-type PMNs (Fig. 2A). To also
analyze whether periodontally relevant pathogens are susceptible
to phagocytotic killing, we isolated PMNs from wild-type and
LAMP-2-deficient mice and incubated these cells with A. actinomycetemcomitans (ratio 1:50) at 37°C for 120 or 240 min, respectively. We also observed that these bacteria were less efficiently
killed by LAMP-2-deficient PMNs (Fig. 2B), and the bacteria were
present intracellularly in a higher number in LAMP-2-deficient
PMNs (Fig. 2, C–E). These data suggest that the phagosomal killing capacity is impaired in LAMP-2 lacking PMNs. To elucidate
whether oxidative or nonoxidative killing pathways were affected
we analyzed the capacity of wild-type and LAMP-2 knockout
PMNs to produce oxygen radicals, which are known to be involved
in bacterial killing by these cells (17, 18). PMNs from both genotypes retained the capacity to react after addition of E. coli or
zymosan by producing similar amounts of oxygen radicals measured by the production of H2O2 (Fig. 3) and reactive oxygen
species (data not shown). These data suggest that impairment of
the nonoxidative, lysosomal killing pathway is mainly responsible
for the reduced killing in LAMP-2-deficient PMNs.
Analysis of LAMP-2-deficient PMNs
Using electron microscopy, we observed that PMNs within the
gingival crevice and PMNs isolated from LAMP-2-deficient mice
were characterized by an accumulation of autophagic vacuoles
(Figs. 1J and 4B), in contrast to wild-type cells (Fig. 4A). Few
early autophagic vacuoles were detected in wild-type PMNs. In
agreement with our earlier results with PMNs in vivo (10), the
LAMP-2-deficient PMNs showed a prominent accumulation of
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We have reported earlier that LAMP-2 deficiency caused an increased postnatal lethality, a reduced weight development, and a
massive accumulation of autophagic vacuoles in numerous tissues
(10, 13). Because we needed to raise the LAMP-2-deficient mice
after the weaning period with liquid food, we wanted to study the
reasons for this impaired feeding behavior. We observed that all
LAMP-2 knockout animals, which were housed in a conventional
breeding facility, exhibited early onset natural periodontitis with
overt migration of molars (Fig. 1, B and C), alveolar bone loss and
furcation involvement (Fig. 1E). Increased mobility of molar teeth
was diagnosed in several animals at the day of sacrifice.
Microscopically, loss of connective tissue attachment level and
alveolar bone crest resorption was already evident at the age of 7
wk after birth (Fig. 1G). In the knockout animals, all molar surfaces exposed to the oral cavity proved to be covered with a layer
of microbial plaque. Because the first molars erupt around day 14,
this response implies that periodontitis had developed within a
time period of 1 mo. Neither plaque nor signs of periodontitis
could be diagnosed in the knockout animals that were supplied
with amoxicillin in the drinking water (Fig. 1M). In none of the
wild-type or other transgenic animals kept in the same conventional breeding facility were signs of periodontitis and plaque development observed.
All interdental areas in the knockout mice exhibited massive
amounts of microbial plaque in the region occlusal to the level of
the gingiva and within the sulcus area (Fig. 1, G and H). However,
neither at the light microscopical level nor at the electron microscopical level were microorganisms observed within the gingival
tissues, except (very occasionally) within the very superficial layers of the sulcular epithelium (Fig. 1I). In the older animals the
biofilm did not only occupy the crevicular domain but had grown
out to cover the free dental surfaces up to the level of the occlusal
plane. Microorganisms were sometimes found within the dentinal
tubules 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 periodontal
pathogens that are commonly found in the human and are associated with natural periodontitis in humans had nested. In particular,
there was no colonization of A. actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, or Tannerella forsytia.
The dominant flora in all animals consisted of facultative anaerobic bacteria with relatively high percentage of Gram-negative rods
with a slightly increased number of Actinomyces species. In histological sections the bacterial morphotypes observed were classified as cocci, rods, and filaments.
The dominant infiltrating cell type in the inflammatory lesions
was the PMN (Fig. 1H). Many of them had infiltrated the junctional and sulcular epithelia. Also within the gingival crevice (outside the gingival tissue) numerous PMNs were observed, many of
them loaded with phagocytosed bacteria (Fig. 1J). Signs of phagocytosis by PMNs or by any other cell type (e.g., macrophage-like
cells) were not found within the tissue, either at the light microscopical level or at the electron microscopical level. No plasma
cells were noted within the connective tissue and only a few cells
belonging to the lymphocytic lineage were identified. The epithelium in the interdental region had lost its normal appearance and
was characterized by extensive proliferation, widened intercellular
spaces, and loss of keratinization (Fig. 1, G and H). Many small
blood vessels were observed throughout the interdental epithelium
477
478
LAMP-2 DEFICIENCY AND PERIODONTAL DISEASE
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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 are
in 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 scans
of 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 interdental
region 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 first
and second mandibular molars in knockout (7 wk). Note proliferation and infiltration by PMNs of the gingival epithelium (H). Accumulation of plaque is
denoted (arrow). I, Dental plaque is depicted at higher magnification. Only in the superficial layers of the gingival epithelium were signs of bacterial
invasion noted (arrowheads). J, Electron micrograph of neutrophil within sulcular area of knockout animal showing electron-lucent vacuoles (v) and
internalized 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 periodontal
ligament (PDL). Note absence of plaque in wild-type animals and high values for plaque in knockout animals (p ⬍ 0.001). A highly statistically significant
difference (p ⬍ 0.001) was found for epithelium and supraalveolar connective tissue. L, Histomorphometric measurements shown mean ⫾ SE in interdental
regions 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 attachment
level (the distance from CEJ to the apical termination of the junctional epithelium) (CEJ-ATJE) in wild-type animals was zero, in knockout groups mean
loss 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-type
groups (p ⬍ 0.005). Molar root length (CEJ-apex) was similar between the two animal groups. M, Effect of antibiotics on development of periodontitis in
one 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 but
also proliferation of epithelium and loss of bone.
The Journal of Immunology
late autophagic vacuoles, containing partially degraded cytoplasmic material (Fig. 4C). Because LC3 was shown to be a specific
marker for early autophagic vacuoles (19), we performed an im-
FIGURE 3. Oxygen radical formation is unaltered in LAMP-2 knockout PMNs. Hydrogen peroxide production by PMNs. After adherence to
surface, PMNs were incubated with opsonized E. coli bacteria or zymosan
for 1 h in phenol red solution containing HRP. H2O2 production was determined from the culture medium. Results represent the mean ⫾ SD of
three experiments.
FIGURE 4. Microscopic examination of LAMP-2 knockout PMNs. A
and B, Electron microscopy of PMNs isolated from the peritoneum of
wild-type (⫹/⫹) and LAMP-2-deficient (⫺/⫺) mice 4 h after thioglycolate
injection. Neutrophil of a 3-mo-old wild-type mouse (A) and LAMP-2deficient (B) mouse are shown. Early autophagic vacuoles (Avi) and late
autophagic 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 detects
autophagic vacuoles in wild-type PMNs (D) and LAMP-2-deficient (E)
PMNs. Scale bar represents 3 ␮m. F, LAMP-2 expression in wild-type
PMNs. Scale bar represents 2 ␮m. Absence of expression in LAMP-2deficient 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-positive
compartments. Scale bar represents 3 ␮m. I and J, Myeloperoxidase staining (MPO) as a marker for primary granules in peritoneal PMNs does not
reveal differences between the two genotypes. Inset, Costaining of MPO
(red) with LAMP-1 (green) showed colocalization in some vesicles in both
wild-type (I) and LAMP-2-deficient (J) PMNs. DAPI was used for counterstaining of nuclei. Scale bar represents 3 ␮m including inset. K and L,
Lactoferrin (LF) staining (green) as a marker for secondary granules. K, In
wild-type PMNs lactoferrin-positive vesicles are found in the center of the
cell and at the membrane. L, LAMP-2-deficient PMNs show aggregated
vesicles and fewer signals at the membrane.
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FIGURE 2. Reduced killing capacity in LAMP-2-deficient PMNs. A,
Scheme of the bacterial killing assay is boxed in upper right. Washing
steps are grayed boxes. A total of 5 ⫻ 105 primary peritoneal PMNs per
well were cultured in a 24-well plate with RPMI 1640 to allow adherence
(A) to plastic surface. Nonadherent cells were removed by washing, and
5 ⫻ 106 E. coli were added. After 1 h of phagocytosis (P), extracellular
bacteria were washed away and subsequently killed by gentamicin (G)
treatment for 30 min. Gentamicin was removed by extensive washing, and
the PMNs were cultured in RPMI 1640 medium containing 0.1% FCS.
After various time points the PMNs were lysed and plated on Luria-Bertani
agar plates incubated at 37°C overnight. CFUs were counted. The experiment 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. actinomycetemcomitans (ratio 1:50) in PBS at 37°C and 5% CO2 for 120 or 240 min, and the
number of CFU was counted. The percentage of viable bacteria in each
sample was then determined by comparing the number of CFU from the
control sample without added neutrophils (100% viability) to the number
of CFU obtained for A. actinomycetemcomitans incubated with neutrophils. Data are presented as mean ⫾ SD. C, Quantitation of the number of
undigested A. actinomycetemcomitans cells inside neutrophils by microscopical examination. Data are presented as mean ⫾ SD. D and E, Phase
contrast pictures of representative images of wild-type (⫹/⫹) (D) and
LAMP-2-deficient (⫺/⫺) (E) neutrophils with nondigested phagocytosed
A. actinomycetmcomitans cells (arrowheads). Scale bar represents 1 ␮m.
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munocytochemical analysis using Abs against this autophagosome
marker. The staining was diffuse in wild-type cells, whereas the
LAMP-2-deficient cells showed a punctuate staining (Fig. 4, D and
E). This result is consistent with autophagic accumulation in
LAMP-2-deficient PMNs.
FIGURE 6. Disturbed acidification in phagosomes in LAMP-2-deficient PMNs. A and B, Peritoneal PMNs were incubated with opsonized
latex beads (3 ␮m) for 1 h, followed by 30 min incubation with 0.1 mM
DAMP, which accumulates in acidified compartments. DAMP was detected with anti-DNP Abs. Phase contrast pictures are shown (inset). DAPI
was used for counter-staining of nuclei. A, Wild-type cells. DAMP-positive
phagosomes are marked (arrowhead). B, LAMP-2-deficient cells. Please
note 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 for
inset image. C, Quantitation of DAMP-positive phagosomes. Results represent mean ⫾ SD of two experiments quantifying 100 phagosomes each
as depicted.
The localization of LAMP-2 in vesicular structures of wild-type
PMNs was easily detectable (Fig. 4F). As expected no LAMP-2
expression was observed in LAMP-2-deficient cells (Fig. 4F, inset). We next analyzed the lysosomal compartment using Abs
against the related major lysosomal membrane protein LAMP-1. In
wild-type PMNs lysosomes were distributed within the entire cytosol and also close to the plasma membrane (Fig. 4G). Importantly, in LAMP-2-deficient PMNs lysosomes appeared enlarged
and clustered in the center of the cell (Fig. 4H). To monitor the
distribution and the presence of primary or secondary granules,
which are essential to supply the phagosome with bactericidal substances (20), we stained PMNs with Abs against myeloperoxidase
(Fig. 4, I and J), a constituent of primary granules, and against
lactoferrin, a constituent of secondary granules (Fig. 4, K and L).
We did not observe differences in the distribution, number and size
of primary granules. Secondary, lactoferrin-containing granules
were less frequently found close to the plasma membrane and
showed a reduced colocalization with LAMP-1 (Fig. 4L) in
LAMP-2 knockout cells, which may suggest an impaired function
and/or transport of these vesicles.
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FIGURE 5. Retarded maturation of latex bead phagosomes in LAMP2-deficient PMNs. Peritoneal PMNs were incubated with opsonized latex
beads (3 ␮m) for 1 h and fixed with 4% paraformaldehyde in PBS. A and
B, Electron microscopy of PMNs with a phagocytosed latex bead from
wild-type (A) and LAMP-2-deficient (B) neutrophil. Note the presence of
both the latex bead phagosome (lb) and autophagic vacuoles (av) in the
LAMP-2-deficient cells. The phagocytic index (PI, beads/cell) was determined by counting at least 250 cells per genotype under the light microscope (phase contrast). The phagocytic index was 2.73 for wild-type and
2.81 for knockout cells. Scale bar represent (A and B) 1 ␮m. C–F, Immunostaining of PMNs with phagocytosed latex beads. Phase contrast pictures
are shown (inset). C and D, Immunohistochemical staining with the
LAMP-1 Ab. DAPI showed a reduced recruitment of LAMP-1 to the latex
bead phagosomal membranes in the LAMP-2-deficient PMNs. Magnification of one phagosome is shown (inset). E and F, Staining with cathepsin
D Ab and DAPI. Scale bar represents 3 ␮m for staining and 5 ␮m for phase
contrast image (inset). Staining also revealed that cathepsin D was less
frequently delivered to LAMP-2-deficient phagosomes. Magnification of
one phagosome is shown (inset). G and H, Recruitment of lysosomal markers as shown in C–F was quantitated for 100 phagosomes in wild-type and
LAMP-2-deficient PMNs in three independent experiments. G, Quantitation of LAMP-1-positive phagosomes. H, Quantitation of cathepsin D
(CathD)-positive phagosomes. Results are shown as mean ⫾ SD.
LAMP-2 DEFICIENCY AND PERIODONTAL DISEASE
The Journal of Immunology
Impaired maturation of phagosomes in LAMP-2-deficient PMNs
Discussion
Lysosomes play a very important role in the oxygen-independent
killing of bacteria, which is believed to be an important mechanism
in the oxygen-deprived periodontal pocket (8). Naturally occurring
periodontal disease normally does not happen in laboratory mice
(21). We now demonstrate that in mice lacking LAMP-2 periodontitis represents a very striking phenomenon. The early onset periodontal disease noted in the present study was clearly related to an
accumulation of plaque. No evidence was found (in any of the
specimens) for invasion of microorganisms into the deeper tissues
of the gingiva. Thus tissue damage and attachment loss were not
likely to be directly caused by bacterial invasion but more likely
the result of inflammation.
We recorded ample evidence of plaque formation in the interproximal areas of the knockout animals. In several cases plaque
had overgrown even the occlusal surfaces. Although our microbiological data were not very detailed, our observations suggest a
mixed 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 same
conditions as their wild-type littermates, in the latter animals bacterial accumulations were never noticed. The prevention of plaque
accumulation and tissue breakdown in the mice receiving antibiotic treatment proves that periodontitis in LAMP-2 knockout mice
is bacteria-related. Our observations also prove that LAMP-2 associated cell functions are pivotal in self-cleaning properties of the
oral cavity. Although we cannot exclude the possibility that the
observed periodontitis was, at least partially, due to systemic effects arising from LAMP-2 deficiency, the most likely reason for
the huge outgrowth of dental plaque in the LAMP-2 knockout
mice 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 associated with severe periodontal destruction (7, 22, 23). Of course
other immune cells might have been affected as well by the deficiency. In this respect, dendritic cells are of interest, which capture
exogenous Ags for eventual processing in endosomes-lysosomes
(24). LAMP-2a facilitates MHC class II presentation of cytoplasmic Ags. Decreased display of cytoplasmic epitopes via class II
molecules was observed in cells with diminished expression of
LAMP-2 (25).
An impaired function of macrophages in bacterial killing in
LAMP-2-deficient mice is unlikely because these cells were only
seldom observed within the affected tissue. We could also recently
show that phagosomal maturation is not affected in LAMP-1- or
LAMP-2-deficient macrophages (14, 26).
PMNs are the most abundant immune cells in the inflammatory
gingivial sites of patients with periodontitis, and their pathogenic
role in this setting has been suggested (27). The role of PMNs in
innate immunity and the specific role of the lysosomal compartment in these cells is underscored by congenital defects such as
Chediak-Higashi syndrome (4) and Papillon-Lefevre syndrome
(28) in which lysosomal secretion events and lysosomal proteolysis is impaired, respectively. Our observations suggest that
LAMP-2-associated functions in PMNs are pivotal in the selfcleaning properties of the oral cavity. They help to orchestrate the
natural defense against oral biofilm formation.
Whereas in LAMP-2-deficient PMNs the distribution and fusion
of primary granules with latex bead phagosomes is apparently unaffected (data not shown), the cellular localization of lactoferrinpositive granules was changed and these granules showed a
reduced colocalization with lysosomal markers, suggesting a disturbed biogenesis, traffic, or function of a subset of granules. Both
types of granules contribute to the killing of bacteria (29) and it is
likely that LAMP-2 contributes to the fusion of granules with
phagosomes.
Phagosome-lysosome fusion is essential for efficient degradation of internalized pathogens. Fusion with lysosomes results
in delivery of an assortment of luminal and membrane proteins
to phagosomes. In a recent study we showed that in LAMP-1
and LAMP-2 double-deficient fibroblasts, phagosomes acquired
the early endosome markers Rab5 and PI3 phosphate, but failed
to recruit Rab7 and did not fuse with lysosomes. We attributed
the deficiency to impaired organellar motility toward the cell
center (14). We proposed that LAMPs might directly or indirectly assist the movement of phagosomes toward the cell center. Elimination of lysosome transport by disruption of microtubules (30) or by interference with RILP or dynein function
(31) impairs contact and fusion between lysosomes and phagosomes. In contrast to fibroblasts, where either LAMP-1 or
LAMP-2 is required for lysosomal motility and phagosomal
maturation, in PMNs the lack of LAMP-2 only seems to be
enough to interfere with the maturation of phagosomes. As already indicated by the accumulation of autophagic vacuoles in
LAMP-2-, but not in LAMP-1-deficient PMNs (32), the lack of
LAMP-2 cannot be compensated by LAMP-1. Similar to the
successful maturation of autophagic vacuoles, the destruction of
phagocytosed bacteria requires the subsequent fusion with early
and late endosomes and lysosomes. We show that the later maturation events are disturbed in LAMP-2-deficient PMNs, leading to impaired bacterial clearance and development of periodontal disease in LAMP-2 knockout mice. Further analyses in
PMNs and LAMP-deficient cells will be required to determine
the molecular mechanisms for the impaired maturation and disturbed lysosomal motility.
In conclusion, our data indicate that LAMP-2 is critically required for the maturation process of phagosomes in PMNs. A lack
of LAMP-2 leads to a reduced maturation and to a reduced bactericidal activity. This functional defect may be directly associated
with the increased susceptibility of LAMP-2 knockout mice to
develop periodontal disease.
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Our data suggested that impaired nonoxidative killing processes
may be the cause for the reduced bacterial killing capacity of
LAMP-2 knockout PMNs. To analyze this further, we made use of
the ability of PMNs to spontaneously phagocytose latex beads in
culture. The ingested latex bead-phagosomes can be easily monitored microscopically (Fig. 5). Electron microscopy confirmed that
LAMP-2-deficient PMNs were capable of phagocytosing such
beads with similar efficiency as wild-type cells (Fig. 5, A and B).
To analyze the maturation defects in more detail, we stained the
cells after 1 h of phagocytosis with proteins localized in the
lysosomal compartment. Whereas the majority of phagosomes
in wild-type cells recruited LAMP-1 (Fig. 5C), and cathepsin D
(Fig. 5E) there was a significant decrease in the number of
LAMP-2-deficient phagosomes positive for either LAMP-1
(Fig. 5D) or cathepsin D (Fig. 5F). Under the given experimental conditions, 60 –70% of wild-type latex bead phagosomes
were positive for lysosomal markers. In contrast, only ⬃30% of
LAMP-2-deficient phagosomes contained lysosomal components (Fig. 5, G and H). In agreement with these results, we also
observed a decreased acidification in the LAMP-2 knockout
phagosomes using immunolabeling of the acidotropic compound DAMP (Fig. 6).
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482
Acknowledgments
We thank Martijn van Steenbergen (University of Amsterdam), Arja Strandell
(University of Helsinki), and Marlies Rusch (University of Kiel) for assistance
in the completion of this project.
Disclosures
The authors have no financial conflict of interest.
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LAMP-2 DEFICIENCY AND PERIODONTAL DISEASE