Clinical Research

C-Reactive Protein Expression Is Up-regulated in Apical

Lesions of Endodontic Origin in Association
with Interleukin-6
Mauricio Garrido, DDS,* Andrea Dezerega, DDS,* Mar
ıa Jos
e Bordagaray, DDS,*
Montserrat Reyes, DDS, MSc,* Rolando Vernal, PhD,* Samantha Melgar-Rodr
ıguez, DDS, MSc,*
P
ıa Ciuchi, DDS,* Rodolfo Paredes, DVM, PhD,† Jocelyn Garc
ıa-Sesnich, Licentiate in Biochemistry,*
Pablo Ahumada-Montalva,* and Marcela Hern
andez, DDS, MSc, PhD*‡

Abstract
Introduction: C-reactive protein (CRP) is the prototype
component of acute-phase proteins induced ultimately
by interleukin (IL)-6 in the liver, but it is unknown
whether periradicular tissues locally express CRP. The
present study aimed to identify whether CRP
messenger RNA synthesis occurs in situ within apical
lesions of endodontic origin (ALEOs) and healthy peri-
odontal ligament and its association with IL-6 and to
determine their protein levels and tissue localization.
Methods: Patients with asymptomatic apical peri-
odontitis and healthy volunteers presenting at the
School of Dentistry, University of Chile, Santiago, Chile,
were enrolled. ALEOs and healthy teeth were obtained
and processed for either immunohistochemistry and
double immunofluorescence to assess IL-6 and CRP
tissue localization, whereas healthy periodontal liga-
ments were processed as controls for real-time
reverse-transcription polymerase chain reaction for
their RNA expression levels and multiplex assay to
determine their protein levels. Statistic analysis was
performed using the unpaired t test or Mann-
Whitney test according to data distribution and Pear-
son correlation. Results: IL-6 and CRP were synthe-
sized in ALEOs, whereas their RNA expression and
protein levels were significantly higher when compared
with healthy periodontal ligament. IL-6 and CRP immu-
nolocalized to the inflammatory cells, vascular endo-
thelial cells, and mesenchymal cells. Both, IL-6 and
CRP colocalized in ALEOs, and a positive correlation
was found between their expression levels (P < .05).
Conclusions: IL-6 and CRP messenger RNA are consti-
tutively expressed in periodontal ligament and up-
regulated in ALEOs along with higher protein levels.
Given their pleiotropic effects, IL-6 and CRP protein
levels in apical tissues might partially explain the deve-
lopment and progression of ALEOs as well as potentially asymptomatic apical
periodontitis–associated systemic low-grade inflammation. (J Endod 2015;41:464–469)
Key Words
Apical periodontitis, C-reactive protein, interleukin-6
A pical periodontitis usually results as a consequence of pulpal infection caused by
bacterial biofilm inside the root canal system of the teeth. The endodontic offenders
and their by-products elicit an immunoinflammatory response that attempts to localize
the infection and prevent further dissemination at the expense of apical periodontal tis-
sue breakdown (1). The result may be the formation of an apical lesion of endodontic
origin (ALEO) that, if initially presented without symptoms, will likely remain asymp-
tomatic (2).
The initiation of inflammatory cascades in ALEOs involves the complex interplay of
multiple cell types, including endothelial cells, polymorphonuclear neutrophils (PMN),
macrophages, lymphocytes, and osteoclasts (1). Nevertheless, the relative composition
of these cellular infiltrates remains controversial, and recent data of our work group
have revealed a high proportion of mast cells (3). These cell populations are known
to produce proinflammatory and osteolytic cytokines, such as interleukin (IL)-1
beta, tumor necrosis factor alpha, and IL-6 (4), leading to the breakdown of apical peri-
odontal tissues, disease development, and progression (5).
IL-6 is a highly pleiotropic cytokine that mediates the host response to injury
and infection and has been observed in apical exudates and human ALEOs (6). IL-6
has potent proinflammatory effects at the local and systemic levels, including the
acute-phase response (7, 8). Ultimately, CRP is produced in response to IL-6 and is
the prototype component of acute-phase proteins. Although the primary source of
IL-6–derived CRP is the liver, extrahepatic synthesis of CRP has been reported. Besides
serum, CRP has recently been detected in gingival crevicular fluid during marginal
chronic periodontitis, but it is unknown whether CRP originates from serum or extra-
hepatic local tissue synthesis (9–11). Extrahepatic CRP has potent proinflammatory
activities, and thus, if present, it might have a role in the local destruction of
periradicular tissues or even the systemic consequences of asymptomatic apical
periodontitis (AAP) (12, 13). In recent years, apical periodontitis has also been
associated with elevated cardiovascular risk through low-grade systemic inflammation
(8, 14). It is unknown whether periodontal tissues and ALEOs express CRP or if this

From the *Laboratorio de Biolog
ıa Periodontal, Facultad de Odontolog
ıa, Universidad de Chile; †Escuela de Medicina Veterinaria, Facultad de Ecolog
ıa y Recursos
Naturales, Universidad Andr
es Bello; and ‡Departmento de Patolog
ıa, Facultad de Odontolog
ıa, Universidad de Chile, Santiago, Chile.
Address requests for reprints to Dr Marcela Hern
andez, Universidad de Chile, Laboratorio de Biolog
ıa Periodontal, Facultad de Odontolog
ıa, Santiago, Chile. E-mail
address: mhernandezrios@gmail.com
0099-2399/$ - see front matter
Copyright ª 2015 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2014.12.021

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protein plays a role in local and systemic consequences derived from
ALEOs. We aimed to identify whether messenger RNA (mRNA) CRP
synthesis occurs in situ within ALEOs and healthy periodontal
ligament and its association with IL-6 and to determine IL-6 and CRP
protein levels and tissue localization.
Methods
Patients presenting at the Clinic of Endodontics, School of
Dentistry, University of Chile, Santiago, Chile, were enrolled if they
had a clinical diagnosis of AAP as previously described (2), including
the presence of at least 1 ALEO (compatible with an apical granuloma
or cyst) detected by apical radiography (>3 mm diameter) caused by
dental caries in teeth with a clinical diagnosis of nonvital pulp. Healthy
teeth were extracted for orthodontic reasons and used as controls.
Exclusion criteria included systemic illness or previous antibiotics
or nonsteroidal anti-inflammatory treatment during the 6-month period
before the study. All the protocols and procedures were approved by the
Ethics Committee from the School of Dentistry of the University of Chile
and were in accordance with the ethical standards of the Declaration of
Helsinki. An informed consent was obtained from all participating indi-
viduals or corresponding forms for their legal guardians in case of un-
derage patients. A total of 42 patients with a diagnosis of AAP and 51
controls were included.
After tooth extraction, ALEOs and healthy teeth were obtained. In
the case of ALEOs, the whole lesions were processed for all the exper-
iments. In the case of control healthy teeth, the whole specimens were
fixed in buffered formaldehyde at 4% for immunohistochemical proce-
dures, whereas healthy periodontal ligaments obtained by surgical sep-
aration from the tooth surface with curettes were processed for DNA
extraction and real-time polymerase chain reaction (PCR) or for tissue
homogenization and multiplexing assay.
Histologic Procedures
Tissue samples were included in paraffin and routine processing
for the diagnosis of apical granuloma (n = 3), radicular cyst (n = 3), or
healthy periapical tissues (n = 3). IL-6 and CRP were immunolocalized
in tissue sections by immunohistochemistry or immunofluorescence.
Immunohistochemistry. To assess IL-6 and CRP immunolocali-
zation in ALEOs and healthy periapical tissues, tissue sections of 4 mm
were cut and deparaffinized, and endogenous peroxidase activity was
quenched by incubation in hydrogen peroxide/methanol. Antigen
retrieval was performed with 0.4% pepsin following manufacturer’s
recommendations (Novocastra Laboratories, Newcastle, UK) and un-
specific blocking was performed with 2.5% horse serum (Kit ABC Uni-
versal Vectastain; Vector Laboratories, Burlingame, CA). Monoclonal
primary antibodies against human IL-6 and CRP (Cat# ab9324 and
#ab32412, respectively; Abcam, Cambridge, MA) were incubated over-
night in 1:8000 and 1:100 dilutions, respectively, and rinsed. The im-
munostaining was performed with Vectastatin Elite ABC kit (Vector
Laboratories) using antimouse and antirabbit biotinylated secondary
antibodies, developed with DAB (Zymed Labs Inc, San Francisco,
CA), counterstained with Mayer hematoxylin (Merck KGaA, Darmstadt,
Germany), and mounted. Slides were examined using an optical micro-
scope (Axiostar Plus; Carl Zeiss, Thornwood, NY), and representative
images were acquired using a microscope-mounted digital camera
(Axiocam ERc5s; Zeiss, G€ottingen, Germany). Positive and negative con-
trols were processed within each series. Positive cells were identified on
the basis of morphologic criteria.
Double Immunofluorescence. To assess whether IL-6 and CRP
colocalized in ALEOs, tissue sections of 3 mm were obtained, cut, and
deparaffinized. Autofluorescence was blocked with glycine 0.1 mol/L
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Clinical Research
for 10 minutes, and unspecific protein blocking was performed with
Pro-Block (ScyTek Laboratories, Logan, UT) following the manufac-
turer’s instructions. The rabbit monoclonal primary antihuman IL-6
and mouse monoclonal antihuman CRP antibodies (Cat# ab9324 and
#ab32412, respectively) were incubated for 1 hour in 1:8000 and
1:250 dilutions, respectively, in Pro-Block solution and rinsed 3 times
with phosphate buffered saline. The slides were subsequently incubated
with appropriate secondary antibodies Alexa488 antirabbit (Invitrogen,
Carlsbad, CA) and Alexa647 antimouse (Abcam), respectively, for 1
hour in 1:500 dilution in Pro-Block; rinsed 3 times with phosphate buff-
ered saline and mounted in Vector Vectashield mounting media con-
taining 40 ,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories);
covered; and sealed. Slides were examined using an epifluorescence
microscope (BX41; Olympus, Center Valley, PA), and representative im-
ages were captured with a microscope-mounted digital camera (Micro-
Publisher 3.3 RTV; QImaging, Surrey, BC, Canada). Digital images were
then processed with image editing software (QCapture Pro v6.0, QImag-
ing).
Real-time Reverse-transcription PCR
To determine IL-6 and CRP mRNA expression levels, ALEOs
(n = 12) and control periodontal ligament samples (n = 12) were sub-
merged in RNAlater (Ambion Inc, Austin, TX) and stored at 4 C for RNA
isolation.
Samples were minced as approximately 1-mm3 fragments and ho-
mogenized in 1 mL TRIzol reagent (Invitrogen Corp, Carlsbad, CA); af-
ter incubation for 5 minutes at room temperature in an RNase-free tube,
200 mL chloroform were added and incubated for 3 minutes at room
temperature. After centrifugation at 12,000g for 15 minutes at 4 C,
the aqueous phase was transferred to a fresh RNase-free tube, and
RNA was precipitated by mixing it with 500 mL isopropyl alcohol incu-
bated for 10 minutes at room temperature and centrifuged at 12,000g
for 10 minutes at 4 C. Then, the RNA precipitate was washed once with
1 mL 75% ethanol and centrifuged at 7000g for 5 minutes at 4 C.
Finally, RNA was resuspended in 30 mL RNase-free Milli-Q water (Merck
Millipore, Darmstadt, Germany), quantified using a spectrophotometer
(Bio-Tek, Winooski, VT), and stored at 80 C at a final concentration
of 1 mg/mL.
Reverse transcription was performed using a First-Strand cDNA
Synthesis Super-Mix kit following the manufacturer’s recommendations
(SuperScript III; Invitrogen, Grand Island, NY). To quantify the mRNA
expression for IL-6 and CRP, 50 ng complementary DNA were amplified
using the appropriate primers (IL-6: F:50 -gcccagctatgaactccttct-30 R:50 -
gaaggcagcaggcaacac-30 and CRP: F:50 -ccagctgtgggtcctgaa-30 R:50 -ca-
cagccccacaaggttc-30 ) designed using the Roche website (https://www.
roche-applied-science.com), and the KAPA SYBR Fast qPCR reagent
(KAPA Biosystems, Woburn, MA) in a StepOnePlus real-time PCR system
(Applied Biosystems, Singapore) as follows: 95 C for 3 minutes fol-
lowed by 40 cycles of 95 C for 3 seconds and 60 C for 30 seconds
and finally a melt curve of 95 C for 15 seconds, 60 C for 1 minute,
and 95 C for 15 seconds for the detection of nonspecific product for-
mation and false-positive amplification. The 18S ribonucleic RNA
expression levels were determined as an endogenous control (18S:
F:50 -ctcaacacgggaaacctcac-30 R:50 -cgctccaccaactaagaacg-30 ).
Tissue Homogenates and Multiplexing Assay
To determine IL-6 and CRP protein levels, after thawing, tissue
samples from ALEOs (n = 36) and control periodontal ligaments
(n = 41) were weighted. Protein extracts were obtained by manual ho-
mogenization in 50 mmol/L Tris-HCl (pH = 7.5), 0.2 mol/L NaCl,
5 mmol/L CaCl2, and 0.01% Triton X-100 (Sigma-Aldrich, St Louis,
C-reactive Protein 465
Clinical Research
MO) buffer adding EDTA-free proteinase inhibitor cocktail (Roche Di-
agnostics GmbH, Mannheim, Germany) in a constant ratio of 10 mL of
buffer per mg of weighted tissue; centrifuged at 10,000g for 5 minutes at
4 C and 1000g for 10 minutes; stored at 80 C until further analysis
with a Milliplex MAP Multiplex assay (Cat #SPR 235 and Cat #SPR236 for
CRP; EMD Millipore, Merck KGaA, Darmstadt, Germany), and read us-
ing a Magpix Luminex platform (Luminex; X-Map Technology, Austin,
TX). Briefly, this quantitative immunoassay is based on the use of
color-coded magnetic microspheres with fluorescent dyes, each coated
with a specific capture antibody. After an analyte is captured by the bead,
a biotinylated detection antibody is added and incubated with streptavi-
din-phycoerythrin (PE) conjugate, the reporter molecule. Then, the mi-
crospheres are allowed to pass through a laser, which excites the
internal dyes marking the microspheres, and a second laser excites
PE, the fluorescent dye on the reporter molecule. Finally, signal proces-
sors identify each individual microsphere and quantify the result based
on the fluorescent reporter’s signals using a standard curve.

Data Analysis
The quantitative reverse-transcription PCR data were analyzed us-
ing the StepOne Software v2.2.2 (Applied Biosystems), and the relative
quantification was obtained by normalizing the IL-6 or CRP mRNA
expression to 18S ribonucleic RNA expression using the 2-DDCt
method. Data were expressed as fold change mean  standard devia-
tion and statistically analyzed using SPSS 15.0 software (Lead Technol-
ogies Inc, Charlotte, NC). The normality of data distribution was
determined using the Kolmogorov-Smirnov test, and differences be-
tween the ALEO group and healthy periodontal ligament controls
were determined using the t test. The correlation coefficient between
IL-6 and CRP was obtained using the Pearson test.
IL-6 and CRP protein were analyzed using STATA v12 software
(StataCorp, College Station, TX) and performing the Mann-Whitney
test. Statistical significance was considered if the P value was <.05.
Results
The study volunteers were composed of 25 females and 26 males
in the control group and 14 females and 28 males in the AAP group
(P = .13). Among them, 45 were nonsmokers and 6 were smokers
in the control group, and 32 were nonsmokers and 10 were smokers
in the AAP group (P = .3). The average age for the control group
was 16.12  5.21 years, and for the ALEO group, it was
48.93  17.68 years (P < .001).
To confirm whether IL-6 and CRP were expressed at the mRNA
level in ALEOs, the mRNA expression for IL-6 and CRP was determined
by quantitative real-time PCR and represented as fold change for each
condition (Fig. 1A). The IL-6 and CRP mRNA expression was detected in
all the samples of both periapical lesions and healthy periodontal liga-
ment used as controls. In periapical lesions, IL-6 was significantly over-
expressed (12.08  9.33, P = .002) compared with healthy controls.
Similarly, CRP (4.088  2.87, P = .003) was also significantly overex-
pressed in periapical lesions compared with healthy controls. The
analyses of correlation yielded significant positive correlations between
IL-6 and CRP in ALEOs (r = 0.88, P < .001; Fig. 1B).
IL-6 protein was detected through multiplexing analysis in only
23.7% of healthy periodontal ligaments and 94.4% of ALEOs
(P < .001). When detected, its levels were low in controls and signifi-
cantly higher in ALEOs (P = .003). CRP was detected in 90.2% of healthy
periodontal ligaments and in 100% of ALEOs (P < .001), and its protein
levels were significantly higher in ALEOs when compared with controls
(P < .001, Table 1).
Figure 1. IL-6 and CRP mRNA expression in ALEOs and controls. (A) The
relative quantification (RQ) of IL-6 and CRP mRNA expression in periapical
lesions and healthy controls, expressed as fold change, was obtained using
the 2DDCt method and by normalizing the mRNA expression to 18S rRNA.
The RQ in periapical lesions was obtained considering the normalized
expression in healthy controls as a reference (RQ = 1). (B) The correlation
of mRNA expression of IL-6 and CRP in periapical lesions. The Pearson cor-
relation coefficient (r) was calculated in 12 periapical lesion samples (cir-
cles). *P < .05.
IL-6 and CRP were identified in ALEOs by immunohistochemistry,
whereas faint to no immunostaining was identified in healthy controls
(Fig. 2A–H). IL-6 and CRP were detected in some mesenchymal fibro-
blastlike cells, endothelial vascular cells, and inflammatory infiltrates
from both apical cysts and granulomas. It was found that immunoreac-
tive IL-6 and CRP were restricted to ALEOs with similar distribution in
both apical granulomas and radicular cysts. Further analysis revealed
that both IL-6 and CRP were found colocalized within ALEO cells
(Fig. 3A–D).
Discussion
Ultimately, CRP is produced in response to IL-6 and is the proto-
type component of acute-phase proteins. Although the primary source
of CRP is the liver, extrahepatic CRP has been reported to have potent
proinflammatory activity. Up to now, only IL-6 has been identified in
ALEOs (10, 13, 15). Our study shows that CRP is constitutively
expressed in periodontal ligaments and is strongly up-regulated in
TABLE 1. Levels of Interleukin (IL)-6 and C-reactive Protein in Apical Lesions
of Endodontic Origin (ALEOs)
Parameter Controls ALEO P value
IL-6 detection (%) 23.68 94.44 <.001
IL-6 levels (pg/mL) 2.34 (1.88) 108.77 (249.85) .003
CRP detection (%) 90.24 100 <.001
CRP levels (ng/mL) 1.02 (1.88) 11.52 (11.68) <.001
Detection of IL-6 (controls, n = 38; ALEOs, n = 36) and CRP (controls, n = 41; ALEOs, n = 36)
expressed as percentages (%). Levels of IL-6 and CRP are expressed as medians (interquartile
range).

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 Clinical Research

Figure 2. IL-6 and CRP immunoexpression in ALEOs and healthy periapical tissues (controls). (A–D) IL-6 and (E–H) CRP. (A and E) Apical granuloma, 40; (B
and F) radicular cyst, 40; (C and G) inflammatory infiltrate, 100; and (D and H) apical periodontium. Arrowheads show immunopositive cells. II, inflammatory
infiltrate; FL, fibroblastlike cells; VE, vascular endothelial cells.

ALEOs in direct association with IL-6 expression levels in resident and
infiltrating inflammatory cells.
We found higher RNA expression and protein levels of IL-6 in ALEOs
compared with healthy periodontal ligaments. In accordance with previous
studies in human apical periodontitis, IL-6 protein levels were significantly
higher in ALEOs compared with healthy controls and in symptomatic apical
periodontitis compared with AAP (6). These results suggest that IL-6 might
be synthesized at low levels in healthy periodontal ligaments but is strongly
up-regulated during apical periodontal inflammation. In fact, the develop-
ment of symptomatic apical abscesses has been associated with the high IL-
6 transcription rate GG genotype and G allele of IL-6 polymorphism in lo-
cus 174 (16). Additionally, IL-6 protein levels were shown to increase
along with ALEO size in apical exudates (17).
In line with previous studies (18), we found IL-6 immunopositive
inflammatory, endothelial, and fibroblastlike cells in ALEOs. Inflamma-
tory radicular cysts and apical granulomas showed similar staining pat-
terns, whereas we confirmed faint to no staining in control healthy
periodontal ligaments. Similarly, a previous study reported up-
regulated IL-6 RNA expression in cultured periodontal ligament fibro-
blasts from chronic periodontitis patients compared with healthy ones.
A faint immunoexpression for the cytokine was also confirmed by
immunofluorescence in the latter group (15).
IL-6 is induced by IL-1 and tumor necrosis factor alpha during
early stages of inflammation, and their effects are synergistic, promoting
the recruitment of PMN and monocytes, shifting from acute to chronic
inflammation, inducing the expression and activation of matrix

Figure 3. Colocalization of IL-6 and CRP in ALEOs. (A) DAPI (nuclei), (B) DAPI + IL-6, (C) DAPI + CRP; and (D) merge, showing cellular colocalization of IL-6
and CRP. Bar: 20 mm.

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Clinical Research
metalloproteinase, and stimulating osteoclastogenesis and bone
resorption (19, 20). Nevertheless, endodontic lesions in IL-6–
deficient mice increased in size compared with wild-type mice (20).
Along with the current finding of IL-6 synthesis in healthy periodontal
ligaments, these results suggest that low basal levels of IL-6 might exert
a physiologic role in periodontal tissues.
CRP is a member of the acute-phase reactants and belongs to the
pentraxin family of proteins from innate immune response. CRP is
induced by proinflammatory cytokines and, ultimately, IL-6 in response
to tissue injury and infection (21), both occurring in ALEOs. Although
the primary source of IL-6–derived CRP is the liver, extrahepatic synthe-
sis of CRP has been reported (12). CRP has recently been detected in
gingival crevicular fluid (9–11). In the present study, we showed that
CRP can be expressed at mRNA levels in situ in both ALEOs and
healthy periodontal ligaments. In line with this, its RNA and protein
levels were significantly higher in ALEO than in healthy periodontal
ligament, suggesting that CRP synthesis might proceed constitutively
in healthy apical periodontium and is strongly up-regulated in response
to endodontic infection and/or apical inflammation. Thus, CRP might be
locally synthesized in periodontal tissues, but we cannot exclude a
serum origin or contribution.
In support of this, CRP mRNA was detected in primary cultures of
healthy periodontal ligament fibroblasts and was significantly up-
regulated in fibroblasts from chronic periodontitis patients (15). On
the other hand, controversy exists regarding the synthesis of CRP
mRNA in gingiva; although protein was consistently detected in gingival
tissues (10, 13), its mRNA was identified only in 1 from the 2 available
reports (13).
As previously described for gingival tissue from periodontitis pa-
tients (13), CRP in ALEOs was localized to inflammatory infiltrates,
vascular endothelial cells, and fibroblastlike cells from apical granu-
lomas and cysts. Considering that the immunostaining patterns were
similar between IL-6 and CRP, we confirmed IL-6 and CRP colocaliza-
tion in ALEOs through double immunofluorescence. Furthermore, we
evidenced a positive correlation between IL-6 and CRP mRNA expres-
sion levels in ALEOs. These findings suggest that IL-6 might act over
target cells in ALEOs by inducing local CRP synthesis. IL-6 classically
acts by binding IL-6 receptor restricted to the cell surface of hepato-
cytes, monocytes, macrophages, and some lymphocytes, whereas in
other cell types IL-6 acts by binding its soluble receptor (12). Further-
more, unpublished data from our group showed that CRP can be syn-
thesized in primary cultures of periodontal ligament fibroblasts in
response to IL-6 stimulation, whereas this effect was abrogated by the
addition of an IL-6 neutralizing antibody. It has been shown that extra-
hepatic CRP has potent proinflammatory activities, and, thus, IL-6–
induced CRP in ALEOs might have a role in the destruction of peri-
odontal tissues (12).
Immunoinflammatory reactions during AAP are not necessarily
restricted to periapical tissues; therefore, antigens and immunoinflam-
matory mediators originating in the root canal system and periapical
areas might leak from vascular inflamed tissues and cause systemic re-
sponses (22). Besides its local effects, IL-6 has potent systemic effects,
stimulating hematopoiesis, T- and B- cell growth and differentiation,
and acute-phase reaction (6), suggesting that ALEOs might cross talk
with systemic inflammation through the IL-6/CRP axis. During recent
years, AP has increasingly been associated with low-grade vascular
inflammation and its systemic cardiovascular consequences, such as
atherogenesis and myocardial infarction (14). CRP is a prototypical
marker of cardiovascular disease because of its strong correlation
and predictive value (23, 24). In fact, high-sensitivity CRP levels
>3 mg/L were defined by the American Heart Association as the cardio-
vascular high-risk category (25). In the present study, levels as high as
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11.52 mg/L were identified in ALEOs. CRP acts locally and systemically
by enhancing inflammation, oxidative stress, and a procoagulant state
(23). Overall, the current results sustain that ALEOs might act as a
continuous reservoir of potent proinflammatory mediators, such as
IL-6 and CRP, that besides inducing local tissue destruction might
also contribute to systemic inflammation. These potential implications
become even more relevant in AAP because of its lack of clinical symp-
toms and unawareness of the patients.
In summary, our study for the first time shows that periodontal lig-
aments constitutively express CRP, which is up-regulated during ALEOs,
in association with IL-6. Still, the regulatory mechanisms and functional
significance in ALEOs and their potentially associated systemic effects
remain to be elucidated.
Acknowledgments
Supported by FONDECYT grant nos. 1090461 and 1120138.
The authors deny any conflicts of interest related to this study.
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