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,
* Division of Dermatology,
Department of Microbiology and Immunology, and
Molecular Biology Institute, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095;
Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan;
¶ Department of Dermatology, Skin Research Center, General Infirmary, University of Leeds, Leeds, United Kingdom; and
|| Genentech, South San Francisco, CA 94080
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation in response to P. acnes. In addition,
peritoneal macrophages from wild-type, TLR6 knockout, and TLR1 knockout
mice, but not TLR2 knockout mice, produced IL-6 in response to
P. acnes. P. acnes also induced
activation of IL-12 p40 promoter activity via TLR2. Furthermore,
P. acnes induced IL-12 and IL-8 protein production by
primary human monocytes and this cytokine production was inhibited by
anti-TLR2 blocking Ab. Finally, in acne lesions, TLR2 was expressed
on the cell surface of macrophages surrounding pilosebaceous follicles.
These data suggest that P. acnes triggers inflammatory
cytokine responses in acne by activation of TLR2. As such, TLR2 may
provide a novel target for treatment of this common skin
disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One of the factors that contributes to the pathogenesis of acne is Propionibacterium acnes, part of normal skin flora that can be significantly increased in the pilosebaceous units of patients with acne (1). Although P. acnes is a Gram-positive bacteria, it is variably and weakly Gram-positive. It is described as diphtheroid or coryneform because it is rod-shaped and slightly curved. A number of unique features of the P. acnes cell wall and outer envelope further distinguishes it from other Gram-positive bacteria. P. acnes synthesizes phosphatidylinositol, this is unlike almost all other bacteria, but is made by virtually all eukaryotes. The peptidoglycan of P. acnes is distinct from most Gram-positive bacteria, containing a cross-linkage region of peptide chains with L, L-diaminopimelic acid and D-alanine in which two glycine residues combine with amino and carboxyl groups of two L, L-diaminopimelic acid residues (2).
P. acnes contributes to the inflammatory nature of acne by
inducing monocytes to secrete proinflammatory cytokines including
TNF-
, IL-1
, and IL-8 (3). In particular, IL-8 along
with other P. acnes-induced chemotactic factors may play an
important role in attracting neutrophils to the pilosebaceous unit. In
addition, P. acnes releases lipases, proteases, and
hyaluronidases which contribute to tissue injury (4, 5, 6, 7).
For these reasons, P. acnes has been a major target of
therapy in inflammatory acne.
The mechanism by which P. acnes activates monocyte cytokine release is unknown but is thought to involve pattern recognition receptors (PRRs)3 of the innate immune system (3). Recently identified Toll-like receptors (TLRs) are one example of PRR. Toll receptors were first identified in Drosophila, and mammalian homologues were found to mediate immune response to microbial ligands (8, 9). Although it has been suggested that TLRs can discriminate between Gram-positive and Gram-negative organisms (10), bacterial ligands from Gram-positive bacteria have been identified that can activate monocytes via TLR2 or TLR4 (11).
The present study was devised to elucidate the mechanism by which P. acnes induces inflammatory cytokines in monocytes. In this study, we provide evidence that P. acnes induces monocyte cytokine production through a TLR2-dependent pathway. The expression of TLR2 in acne lesions indicates that activation of TLR2 can contribute to inflammation at the site of disease activity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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P. acnes was obtained from American Type Culture Collection (Manassas, VA) and prepared by probe sonication. The level of endotoxin contaminating the P. acnes was quantified with a Limulus Amoebocyte Lysate assay (BioWhittaker, Walkersville, MD) and found to be <0.1 ng/ml. LPS derived from Salmonella typhosa (Sigma-Aldrich, St. Louis, MO) and the 19-kDa lipoprotein of Mycobacterium tuberculosis (12) were used. The following Abs were used: a mAb specific to human TLR2 (clone 2392; Ref. 13) and TLR4 (clone HTA125; Ref. 14) was provided by P. J. Godowski (Genentech, San Francisco, CA); B355.1 (anti-CD3; Biomeda, Foster City, CA), RPA-MI (anti-CD14; Zymed Laboratories, South San Francisco, CA), NA1/34 (anti-CD1a; DAKO, Carpinteria, CA), and IgG controls (Sigma-Aldrich).
Samples from patients
Patients were clinically diagnosed with acne at the General Infirmary at Leeds (Leeds, U.K.). After informed consent was obtained, comedones and inflamed acne lesions (papules and pustules) from the upper back were biopsied under local anesthesia using a 4-mm punch. Nineteen acne samples (3 comedones, 1 pustule, and 15 papules) were obtained from sixteen different patients. To ascertain the duration of the papules, an established "mapping" technique (15) was used which allowed a reasonably accurate assessment of the duration of the lesion. Such timed biopsies were classified into four time zones (up to 6 h, from 6 to 24 h, from 24 to 48 h, and from 48 to 72 h). The biopsies were snap frozen in liquid nitrogen and stored at -70°C until sectioning.
NF-B activation in human TLR-transfected cell lines
TLR2 negative human embryonic kidney (HEK) 293 cells were stably
transfected with TLR2 and CD14 (9). Cells were plated at
1 x 105 cells/well in six-well plates and
transiently transfected the following day with the NF-B responsive
endothelial leukocyte adhesion molecule (ELAM) enhancer
luciferase (pGL3) reporter gene (0.5 µg/ml) by the Superfect
protocol at a 1:3 ratio of DNA to Superfect (Qiagen, Valencia, CA).
Multiple transfectants were pooled and divided for activation with
P. acnes, M. tuberculosis 19-kDa lipoprotein, or
LPS for 6 h, lysed in reporter lysis buffer (Promega, Madison,
WI), and used in the luciferase assay. BaF3 cells stably expressing
TLR4, MD2, CD14, and an ELAM luciferase reporter gene (14)
were plated at 7.5 x 105 cells/well and
activated with P. acnes, M. tuberculosis 19-kDa
lipoprotein, or LPS. Cells were harvested 6 h after activation and
used in the luciferase assay.
Responses in TLR-deficient mice
Peritoneal macrophages from TLR2-/- (11), TLR6-/- (16), and TLR1-/- (17) mice were collected 3 days after i.p. injection of 2 ml of 4% thioglycollate (Difco, Detroit, MI) and cultured in RPMI 1640 medium supplemented with 10% FCS. Cells (5 x 104) were incubated in the presence of the indicated concentration of P. acnes for 24 h. Concentrations of IL-6 in the culture supernatants were measured by ELISA (R&D Systems, Minneapolis, MN). The data represent the mean ± SD of triplicate wells.
IL-12 p40 promoter activity
The murine macrophage cell line, RAW 264.7 (American Type
Culture Collection), was transiently transfected with a murine IL-12
p40 promoter chloramphenicol acetyltransferase (CAT) reporter as
previously described (18). TLR2 dominant negative mutant
(TLR2 dn1) expression plasmids were transfected together with the IL-12
p40 promoter construct and -galactosidase as an internal control.
Transfected cells were either left unactivated or stimulated with
P. acnes or M. tuberculosis 19-kDa lipoprotein
for 24 h. IL-12 p40 promoter activity was measured according to
CAT activity (percent chloramphenicol acetylation) with a phosphor
imager (Amersham, Sunnyvale, CA). Data were normalized to a
cotransfected -galactosidase construct for transfection
efficiency.
Cytokine ELISA
PBMCs were isolated from normal healthy volunteers on Ficoll-Paque gradients (Pharmacia, Piscataway, NJ) and cultured for 1 h in 1% human serum. Adherent cells were recovered and plated (1 x 104 to 5 x 104/well) in 96-well plates. Cells were left untreated or incubated with mouse anti-human TLR2 neutralizing mAb, mouse anti-human TLR4 neutralizing Ab, or with isotype control mouse Abs, IgG1 and IgG2a, for 30 min before stimulation with LPS (10 ng/ml), P. acnes (1:100), or 19-kDa lipoprotein (50 ng/ml). Supernatants were harvested 18 h later and assayed for IL-12 p40 and IL-8 by ELISA (BD PharMingen, San Diego, CA). All samples were assayed in duplicate.
Immunoperoxidase staining
Cryostat sections (3–4 µm) were acetone fixed and blocked with normal horse serum before incubation with the mAbs for 60 min, followed by biotinylated horse anti-mouse IgG for 30 min. Primary Abs were visualized with the ABC Elite system (Vector Laboratories, Burlingame, CA), counterstained with hematoxylin, and mounted in aqueous dry mounting medium (Crystal Mount; Biomeda).
Two-color immunofluorescence labeling in acne lesions
Cryostat sections (3–4 µm) were fixed in acetone and blocked with 10% goat serum for 30 min. Double immunofluorescence was performed by serially incubating sections with mouse anti-human CD14 or CD3 mAbs for 1 h followed by incubation with isotype-specific FITC-conjugated goat anti-mouse IgG2a (Caltag Laboratories, Burlingame, CA). Sections were then incubated with anti-TLR2 (1 µg/ml) for 1 h, followed by a tetramethylrhodamine isothiocyanate-conjugated anti-mouse IgG1 (Southern Biotechnology Associates, Birmingham, AL). Sections were mounted in Vectashield mounting medium (Vector Laboratories). Controls included isotype-matched irrelevant Abs.
Confocal laser microscopy
Double immunofluorescence of sections and cells was examined with a Leica-TCS-SP inverted confocal laser-scanning microscope (Heidelberg, Germany) and illuminated with 488 and 568 nm of light. Images decorated with FITC and tetramethylrhodamine isothiocyanate were recorded simultaneously through separate optical detectors with a 530-nm band-pass filter and a 590-nm long-pass filter, respectively. Pairs of images were superimposed for colocalization analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have demonstrated that TLR2 mediates the response
of several ligands from Gram-positive organisms. Therefore, we sought
to determine whether TLR2 is sufficient for P. acnes-induced
gene activation. HEK 293 cells and BaF3 cells were used because these
cells do not express endogenous TLRs and are unresponsive to microbial
ligands (12, 14). HEK 293 cells (expressing TLR2, CD14,
and a NF-B responsive ELAM enhancer) and BaF3 cells (expressing
TLR4, CD14, MD2, and ELAM) were activated with P. acnes, M.
tuberculosis 19-kDa lipoprotein, or LPS. NF-B activation was
examined because it has been shown that NF-B activation is required
for several proinflammatory cytokine promoter activities
(18, 19, 20, 21). In stable transfectants expressing TLR2 and
CD14, P. acnes induced NF-B activation (Fig. 1
a). In contrast, P.
acnes could not activate NF-B in transfectants expressing TLR4,
CD14, and MD2. Similar results were seen when cells were stimulated
with 19-kDa lipoprotein from M. tuberculosis. In contrast,
LPS activated NF-B in stable transfectants expressing TLR4 but not
TLR2. This is consistent with findings that LPS activation of cytokine
production in monocytes is dependent on TLR4.
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b). P.
acnes also induced IL-6 release from
TLR1-/- mice (Fig. 1c). As controls,
the diacylated lipopeptide mycoplasmal macrophage-activating
lipopeptide-2 kDa activated wild-type and
TLR1-/- mice, but not
TLR2-/- or TLR6-/-
mice, whereas LPS activated macrophages from all mice (data not shown).
In contrast, a synthetic
N-palmitoyl-S-dipalmitoylglycerl
(Pam3) CSK4 activated
wild-type mice, but TLR1-/- activation was
partially inhibited as previously described (Ref. 17 ; data
not shown). These results indicate the specificity of the response that
TLR2 but not TLR4, TLR6, or TLR1 mediates P. acnes-induced
cell activation. P. acnes induces IL-12 p40 promoter activity via TLR2
IL-12 is a pivotal cytokine in activating Th1 T cell responses and is one of the major proinflammatory cytokines produced by monocytes in response to Gram-positive organisms. To determine whether P. acnes-induced IL-12 promoter activity, an IL-12 p40 promoter CAT reporter construct was transiently transfected into the murine macrophage cell line RAW 264.7. Cells were stimulated with P. acnes and the promoter activity was measured by CAT assay. P. acnes induced IL-12 p40 promoter activity in a dose-dependent manner and at a level comparable to M. tuberculosis 19-kDa lipoprotein (data not shown).
To determine whether P. acnes-induced cytokine production
was dependent upon TLR activation, the RAW 264.7 macrophage cell line
was transfected with the TLR2 dn1 construct containing a truncation of
13 amino acids at the COOH terminus, along with the IL-12 p40 promoter
construct. P. acnes induced IL-12 p40 promoter activity in
the absence of TLR2 dn1 in RAW cells, but this activity was abrogated
in cells transfected with the TLR2 dn1 construct (Fig. 2). Consistent with previously published
results, transfection of TLR2 dn1 also inhibited M.
tuberculosis 19-kDa lipoprotein-induced IL-12 p40 promoter
activation (12). The IL-12 p40 promoter activity was not
inhibited by transfection of RAW cells with another control vector
containing the IL-1R (data not shown). These data suggest that P.
acnes activates IL-12 p40 promoter activity in a TLR2-dependent
mechanism.
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We next determined whether cytokine protein production was also
dependent on TLR activation. Primary human monocytes from normal donors
were stimulated with various dilutions of P. acnes sonicate
and cytokine production was measured. IL-12 was measured given that
IL-12 promoter activation by P. acnes occurred via TLR2. We
found that P. acnes induced IL-12 production by monocytes in
a dose-dependent manner (Fig. 3a). P. acnes also
induced the release of IL-8, a cytokine involved in neutrophil
chemotaxis (Fig. 3b). These findings were consistent in all
normal donors tested (n = 3).
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65% with the addition
of anti-TLR2 Ab to the culture (Fig. 3c). In contrast,
the addition of anti-TLR4 and isotype control Abs had no
significant effect on IL-12 production by human monocytes, suggesting
that the blocking effect was specific to TLR2. Induction of IL-12
production by monocytes upon addition of another Gram-positive
bacterial ligand, the M. tuberculosis 19-kDa lipoprotein,
was also blocked with the addition of anti-TLR2 (data not shown),
as demonstrated previously (12). Similarly, the production
of IL-8 was also blocked by 50% by the addition of anti-TLR2 Ab
(Fig. 3d). Anti-TLR4 and isotype control Abs had no
significant effect on IL-8 production. These results suggest that one
mechanism by which P. acnes induces IL-12 and IL-8
production in human monocytes is by the activation of TLR2. TLR2 is expressed on macrophages in acne lesions
We next wanted to determine whether there was any evidence for the
TLR-dependent mechanism occurring at the site of the disease activity.
We obtained acne biopsies from patients and analyzed the lesions for
TLR expression. Immunohistochemistry labeling using a mAb
specific to TLR2 (23) revealed TLR2 expression on large
ovoid cells within acne lesions (Fig. 4).
TLR2+ cells were detected primarily in the
inflammatory infiltrate around the perifollicular/peribulbar region.
Numerous CD14+ and CD3+
cells were detected in the similar area. We also detected a number of
CD1a+ cells, but they were within the follicular
wall and in the epidermis. Anti-CD20 and control Abs did not stain any
of the cells (data not shown). All acne lesions tested
(n = 19) contained TLR2+ cells
whether the tissue was obtained from comedonal, papular, or
pustular lesions. TLR2+ cells were not
detected in normal skin biopsies (data not shown).
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). In lesions obtained between 12 and
24 h, TLR2+ cells were found to be more
numerous around the pilosebaceous follicles. Finally, in older lesions
obtained between 48 and 72 h, even greater numbers of
TLR2+ cells were detected. From these
experiments, we concluded that the infiltration of
TLR2+ cells was an early event in the evolution
of acne lesions. Furthermore, the frequency of cells expressing TLR2
proteins is up-regulated during the inflammatory process up to 48–72 h
after the onset of acne lesions.
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a). In contrast, although
CD3+ cells were abundant in acne lesions, CD3 did
not colocalize with TLR2 (Fig. 6b). These data suggest that
TLR2 is expressed on cells of the monocyte/macrophage lineage in acne
lesions.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Toll receptors were first identified in Drosophila as an
integral part of the innate immune system and have been shown to play a
crucial role in antimicrobial defense in adult flies (24, 25). Recent studies suggest that mammalian Toll homologues,
TLRs, mediate responsiveness to a variety of molecular structures from
microbial pathogens. The present study provides evidence that TLR2
mediates innate immune responses to P. acnes. P. acnes
activated NF-B in cell lines transfected with TLR2 but not TLR4. In
addition, peritoneal macrophages from wild-type,
TLR6-/-, and TLR1-/-,
but not TLR2-/-, mice produced IL-6 in response
to P. acnes. A role for TLR2 in mediating the response to
P. acnes was further demonstrated by using a dominant
negative construct of TLR2 that inhibited P. acnes induction
of cytokine promoter activity. TLR2 mediated the ability of P.
acnes sonicate to activate monocyte-release of IL-12 and IL-8,
because cytokine induction could be blocked using an anti-TLR2 mAb.
Using transgenic mice, Akira and colleagues (16, 17) have
demonstrated that TLR1 associates with TLR2 and recognizes triacylated
lipopeptide, but TLR6 and TLR2 interact to recognize diacylated
lipopeptide. The peptidoglycan of P. acnes is distinct from
most Gram-positive bacteria, containing a cross-linkage region of
peptide chains with L,
L-diaminopimelic acid and
D-alanine in which two glycine residues combine
with amino and carboxyl groups of two L,
L-diaminopimelic acid residues (2).
Previous studies have indicated that the addition of jimson lectin,
which binds peptidoglycan, blocked the ability of P. acnes
to induce cytokines by 70% (3). Given our result that
P. acnes induced IL-6 release from macrophages from
wild-type, TLR6-/-, and
TLR1-/- mice, but not from
TLR2-/- mice, it is likely that the TLR ligand
in P. acnes is a peptidoglycan. Further studies will be
required to identify the TLR2 ligands present in P. acnes
and their role in inflammation.
The primary event in inflammatory acne involves the disruption of the follicular epithelium and colonization of the follicles with P. acnes with subsequent inflammatory reactions in the surrounding dermis. The detection of TLR2+ cells in the perifollicular region provides indirect evidence that TLR2 activation contributes to the pathogenesis of acne, suggesting that these cells promote inflammatory responses at the site of the disease activity. This disease mechanism was supported by the colocalization of TLR2 with CD14, indicating its presence on cells of the monocyte/macrophage lineage. Previously, TLR2+ cells have been demonstrated in tuberculoid lesions (26) but not all macrophages in lesions express TLR2, for example those in lepromatous leprosy (our unpublished observations). Furthermore, activation of TLR2 on monocytes releases proinflammatory cytokines, IL-12 and IL-8. IL-8 attracts neutrophils to the site of active lesion, and release of lysosomal enzymes by neutrophils leads to rupture of follicular epithelium and further inflammation (27). In contrast, IL-12 promotes development of Th1-mediated immune responses. Overproduction of Th1 cytokines such as IL-12 has been implicated in the development of tissue injury in certain autoimmune and inflammatory diseases (28, 29, 30, 31, 32, 33, 34). In this manner, the activation of TLR2 on monocytes and other TLRs as well as other inflammatory cells are likely involved in the pathogenesis of acne.
In addition to its primary role in combating infection, the immune
system also plays a role in the pathogenesis of certain disease states.
In fact, the very pathogens that the immune system is attempting to
fight often play a critical role in mediating the inflammatory
responses that lead to disease states. Examples of this include group A
-hemolytic streptococcus in rheumatic fever, rheumatic heart
disease, and glomerulonephritis, Helicobacter pylori in
gastritis and peptic ulcer disease, Chlamydia
pneumoniae in atherosclerosis, and Pityrosporum
ovale in seborrheic dermatitis. In all of the above examples,
infection by the organism itself is not the main cause of the disease,
but rather the various inflammatory responses initiated by the
microbial agents lead to the destruction of the host tissue. Such
responses include the formation of immune complexes, the recruitment
and activation of neutrophils and monocytes, the release of cytokines,
and the release of degradative enzymes. P. acnes has been
implicated as an important mediator of inflammation in the pathogenesis
of acne. Clearly, treatment of patients with antibiotics reduces the
number of P. acnes and inflammatory cells and results in
clinical improvement of acne lesions (35, 36, 37).
Interestingly, the inflammatory cytokine responses triggered by
P. acnes and mediated by TLR2 are unlikely to have a
protective role in acne. It is tempting to speculate that the release
of proinflammatory cytokines mediated through TLR2 has a harmful effect
in acne by promoting inflammation and tissue destruction. Given these
data, TLR2 is a logical target for therapeutic intervention to block
inflammatory cytokine responses in acne and other inflammatory
conditions in which tissue injury is detrimental to the host.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robert L. Modlin, Division of Dermatology, University of California, Los Angeles, School of Medicine, 52-121 Center for Health Sciences, 10833 Le Conte Avenue, Los Angeles, CA 90095. E-mail address: rmodlin{at}mednet.ucla.edu
3 Abbreviations used in this paper: PRR, pattern recognition receptor; TLR, Toll-like receptor; ELAM, endothelial leukocyte adhesion molecule; HEK, human embryonic kidney; CAT, chloramphenicol acetyltransferase; TLR2 dn1, TLR2 dominant negative mutant.
Received for publication December 6, 2001. Accepted for publication May 17, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B half-site. Mol. Cell. Biol. 15:5258.[Abstract]
B subunit-specific regulation of the interleukin-8 promoter. Mol. Cell. Biol. 13:6137.B. Mol. Cell. Biol. 13:7191.
(IFN-), IL-10, IL-12 and transforming growth factor- (TGF-) mRNA in synovial fluid cells from patients in the early and late phases of rheumatoid arthritis (RA). Clin. Exp. Immunol. 103:357.[Medline]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), or with a vector control (
). Transfected cells were
stimulated with P. acnes sonicate or M.
tuberculosis 19-kDa lipoprotein, or left unstimulated (
) for
24 h. Activation of IL-12 p40 promoter activity was measured
according to CAT activity (percent chloramphenicol acetylation) with a
phosphor imager. Data reflects at least two independent experiments and
are reported as a percentage of Ag-stimulated IL-12 p40 promoter
activity cotransfected with a vector control. Media controls were
comparable between vector control and TLR2 dn1 transfectants.
