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Fearing Research Laboratory, Department of Obstetrics and Gynecology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115;
Department of Infectious Diseases, Boston Medical Center, Boston University School of Medicine, Boston, MA 02118; and
Institute of Cancer Research and Molecular Biology, Norwegian University of Science and Technology, Trondheim, Norway
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation and proinflammatory cytokine production. The
presence of soluble CD14, a high-affinity receptor for LPS and other
bacterial ligands, enhanced the sensitivity of genital tract epithelial
cells to both low and high concentrations of bacteria, suggesting that
soluble CD14 can act as a coreceptor for non-TLR4 ligands. These data
demonstrate that the response to N. gonorrhoeae and
other Gram-negative bacteria at the mucosal surface of the female
genital tract occurs in the absence of endotoxin recognition and
TLR4-mediated signaling. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The epithelial cells that line mucosal surfaces are often the first cells to contact microbial pathogens, as normally there are very few immune cells present in the cervicovaginal mucosa and lumen (6, 7). Here, they initiate and coordinate the inflammatory response, alerting adjacent epithelium and the underlying immune cells of the potential danger posed by various microorganisms. Despite the important role that the genital tract mucosa plays in immune defense, little is known about the mechanism of epithelial cell activation by pathogens and the receptors and secondary mediators involved in this regulated response. We have recently established an in vitro model to study epithelial-microbial interaction in this compartment using immortalized epithelial cells derived from human vagina, ectocervix, and endocervix. These cells maintain the normal immunobiological characteristics of their tissues of origin and differ extensively from commonly used tumorigenic cell lines, such as HeLa cells (8, 9). We have used this model to study the downstream effects of cell signaling following Neisseria gonorrhoeae infection. Our previous investigations showed that all three epithelial cell lines respond to N. gonorrhoeae by up-regulation of cytokines and adhesion molecules and that these responses are independent of bacterial internalization and IL-1 production (10). However, the key components of the signaling pathways involved in the inflammatory responses of cervicovaginal epithelial cells to Gram-negative bacteria have yet to be identified.
In this study we determined the expression profiles of members of the Toll-like receptor (TLR)3 family in human cervicovaginal epithelial cells and correlated this with proinflammatory responses to bacterial pathogens. The TLR family plays a pivotal role in the recognition of bacterial ligands by the innate immune system (11, 12). Toll was initially identified as a receptor involved in embryonic development, where it controls dorsoventral polarization (13). It was later demonstrated that Toll and the related molecule, 18-Wheeler, control important antimicrobial responses against both fungi and bacteria in the adult fly (14). At least 10 mammalian orthologs of Toll, designated TLRs, have been identified, and several of them have been implicated in cellular responses to bacterial pathogens. While both TLR2 and TLR4 were initially implicated in cellular responses to LPS, the overwhelming evidence to date suggests that these two receptors have different roles in the recognition of pathogens. TLR4 is required for recognition of LPS (also known as endotoxin), the most proinflammatory cell wall component of Gram-negative bacteria (15, 16, 17, 18, 19). A small secreted protein, known as MD2, which associates with TLR4 on the cell surface of innate immune cells, may also be required for TLR4-mediated responses to LPS (20, 21). TLR2 has a broader role as a pattern recognition receptor for a variety of microbes and Gram-positive ligands, e.g., bacterial lipopeptides, lipoprotein, lipoteichoic acid, and peptidoglycan (22, 23, 24, 25, 26, 27, 28, 29). There is also some evidence that TLR2 may require association with TLR6 for recognition of certain ligands, including some lipoproteins (30, 31), peptidoglycan (32), and phenol-soluble modulin (33). In addition, TLR9 has been associated with responses to bacterial DNA (34), and TLR5 has been associated with responses to bacterial flagellin (35).
A key component of both TLR and IL-1 signaling is the adapter molecule
MyD88. MyD88 is a unique protein that has homology to the cytoplasmic
domain of the IL-1 and Toll receptors at the C-terminal, and an
N-terminal death domain (36, 37). It is recruited by the
IL-1 or Toll receptor complex after receptor stimulation and is an
essential step in the sequential recruitment of the downstream kinases
IL-1R-associated kinase and TNFR-associated factor-6, activation of
NF-B, and transcription of a myriad of proinflammatory molecules
(38, 39).
We found that cervical and vaginal epithelial cells expressed mRNA for
TLR1, -2, -3, -5, and -6, but failed to express TLR4 and MD2, two
essential components of the receptor complex for responses to LPS.
Consistent with this, cervical epithelial cells failed to respond to
protein-free preparations of endotoxin derived from enteric
(Escherichia coli) and nonenteric (N.
gonorrhoeae) Gram-negative species, as measured by cytokine
production and NF-B activation. In contrast, the cells remained
sensitive, in a MyD88-dependent fashion, to TLR2 bacterial ligands,
i.e., lipoproteins and peptidoglycan, as well as to bacterial lysates
and infection with whole N. gonorrhoeae. Interestingly,
although cervicovaginal cells lack endogenous membrane CD14, soluble
CD14 (sCD14), which was found in semen and cervicovaginal secretions,
enhanced their proinflammatory host response to N.
gonorrhoeae. These findings suggest that the lower female genital
tract responds to Gram-negative bacterial components in the absence of
endotoxin recognition and TLR4-mediated signaling.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBS and trypsin-versene mixture (trypsin-EDTA) were obtained
from BioWhittaker (Walkersville, MD), and FCS (<10 pg/ml LPS) was
purchased from Summit Biotechnology (Greeley, CO). Recombinant human
IL-1
and TNF-
were purchased from R&D Systems (Minneapolis, MN).
Recombinant human sCD14 and LPS binding protein (LBP) were gifts from
H. Lichenstein (Amgen, Thousand Oaks, CA). Lipo-oligosaccharide (LOS)
purified from N. gonorrhoeae (strain 1291) was a gift from
N. Qureshi (University of Wisconsin, Madison, WI). LPS purified from
E. coli K235 was purchased from List Biologicals (Campbell,
CA). Contaminating endotoxin proteins were removed by phenol
re-extraction as described previously (40, 41). Synthetic
lipopeptide corresponding to the N termini of the Treponema
pallidum 47-kDa lipoprotein (designated 47-L) was a gift from J.
Radolf (University of Connecticut, Farmington, CT). Synthetic
lipopeptide based on the full-length MALP-2 membrane lipopeptide from
Mycoplasma fermentans (sMALP-2) was a gift from G. Rawadi
(Institut Pasteur, Paris, France) (27, 42). Soluble
peptidoglycan (designated sPG) was a gift from R. Dziarski (Indiana
University School of Medicine, Gary, IN). Heat-killed Listeria
monocytogenes (HKLM) was a gift from G. Teti (Policlinico
Universitario, Messina, Italy). Ab to human TLR2, TL2.1, was a gift
from T. Espevik (Norwegian University of Science and Technology,
Trondheim, Norway). Ab to human TLR4, HTA125, was a gift from K. Miyake
(Saga Medical School, Saga, Japan). PE-conjugated sheep anti-mouse
IgG (Sigma-Aldrich, St. Louis, MO) was used for Ab detection in flow
cytometry. In some cases Ab preparations were also directly labeled
with Alexa Fluor 488 using a commercially available kit (Molecular
Probes, Eugene, OR). A human bone marrow cDNA library was purchased
from Invitrogen (Carlsbad, CA).
Bacteria and bacterial lysates
N. gonorrhoeae laboratory strain 1291 was grown overnight on chocolate agar. Colonies were scraped and resuspended in PBS with 0.1% BSA. Cell density was adjusted to an OD600 equal to 0.1, corresponding to 108 CFU/ml bacteria. Crude bacterial lysates were generated from bacteria resuspended in pyrogen-free, sterile distilled water, subjected to a freeze-thaw cycle, and vortexed vigorously. Lysates were treated with DNase for 1 h, stored at -20°C, and vortexed before use.
Cell lines
Generation of the HPV16/E6E7 immortalized endocervical (End1/E6E7), ectocervical (Ect1/E6E7), and vaginal (Vk2/E6E7) epithelial cell lines has been previously described (8). The vaginal epithelial cells were derived from a different donor than the endocervical and ectocervical cells. Cells were maintained in keratinocyte serum-free medium (Life Technologies, Gaithersburg, MD) supplemented with 50 µg/ml bovine pituitary extract and 0.1 ng/ml epidermal growth factor (both supplied by the manufacturer), 10 µg/ml ciprofloxacin (Miles Pharmaceuticals, West Haven, CT), and CaCl2 to a final concentration of 0.4 mM. Cells tested negative for mycoplasma by PCR (43). HMEC-1 (human microvascular endothelial cells) were a gift from M. Arditi (Cedars Sinai Medical Center, Los Angeles, CA) (44). CHO-K1 fibroblasts stably transfected with human TLR2 or TLR4 were a gift from D. Golenbock (University of Massachusetts Medical Center, Worcester, MA) (27). Primary endocervical and ectocervical epithelial cells were purchased from BioWhittaker/Clonetics (San Diego, CA) and maintained according to the manufacturer’s instructions.
RT-PCR analysis
Total RNA was isolated from cells using Tri-Reagent according to
the manufacturer’s instructions (Molecular Research Center,
Cincinnati, OH) and was treated with RQ1 RNase-free DNase (Promega,
Madison, WI). Reverse transcription and PCR were conducted using
Superscript II (Life Technologies) and Taq DNA polymerase
(Promega), respectively. The PCRs were conducted for 25 cycles in a
PerkinElmer GeneAmp 2400 PCR machine (PerkinElmer/Cetus, Norwalk, CT)
using the primer pairs and conditions described in Table I
. Lack of DNA contamination in the RNA
preparations was confirmed by PCR in the absence of reverse
transcription.
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Cells were grown in 96-well tissue culture dishes at a density of 105 cells/well and stimulated for the stated period of time. Plates were centrifuged at 400 x g for 5 min at 4°C before cell culture supernatants were collected. Samples were stored at -80°C and later assayed for IL-8 and IL-6 using PeliKine compact ELISA kits from Research Diagnostics (Flanders, NJ) using the manufacturer’s protocol. OD was measured using a Bio-Kinetics microplate reader (Bio-Tek Instruments, Winooski, VT). All cytokine assays were plated in triplicate, and experiments were repeated at least three times. Data are reported as the mean of triplicate samples ± SD. Significance (p value) was calculated using a t test.
Nuclear extracts and NF-B translocation assay
One day before stimulation cells were grown in six-well tissue
culture dishes at a density of 1 x
106/well. After a 60-min stimulation cells were
harvested and nuclear extracts were prepared as previously described
(45). Nuclear NF-B was identified using EMSA with a
32P-labeled oligonucleotides containing the
consensus sequence for NF-B binding from the murine Ig L chain
gene enhancer (45).
Expression plasmids
The following expression plasmids were gifts from D. Golenbock
(University of Massachusetts Medical Center): reporter plasmid
pELAM-luc, which transcribes firefly luciferase from an
NF-B-dependent promoter (16); mutant murine MyD88
dominant-negative (MyD88 DN), containing the C-terminal half (aa
146–296) of the protein in the mammalian expression vector pLEP3
(46); and mutant murine TLR2 dominant-negative, which
lacks the terminal 13 aa of the TLR2-coding sequence, expressed in the
mammalian expression vector pcDNA3 (46). The expression
plasmid for nontagged human TLR4 in the mammalian expression plasmid
pcDNA3 was a gift from R. Medzhitov (Yale University, New Haven, CT)
(12). The expression plasmid for human MD2 in the
mammalian expression plasmid pEFBOS was a gift from K. Miyake (Saga
Medical School) (20). All plasmids were prepared using
EndoFree Plasmid Maxi Kit plasmid DNA purification columns from Qiagen
(Valencia, CA).
Transient transfection and NF-B luciferase reporter assay
Transient transfections were conducted using either DEAE-dextran or a lipid transfection reagent. For the DEAE-dextran method, cells were plated at a density of 2.5 x 105 cells/well and transfected with 0.25 µg DNA using DEAE-dextran (47). For the lipid transfection, cells were plated at a density of 104/well and transfected with 0.1 µg DNA using Effectene (Qiagen). Cell activation was determined by measuring luciferase activity in the total cellular lysate using an assay kit from Promega according to the manufacturer’s instructions and a Monolight 3010 luminometer (BD PharMingen, San Diego, CA). All transfection experiments were performed in triplicate and repeated at least three times. Data are reported as the mean of triplicate samples ± SD. Significance (p value) was calculated using a t test.
Flow cytometric analysis
Cells growing in tissue culture were harvested with 1 mM EDTA and incubated for 30 min on ice with Alexa-conjugated anti-TLR2 or anti-TLR4 Ab or with control mouse IgG at a concentration of 20 µg/ml. After labeling, the cells were washed, resuspended in PBS/1% FBS, and analyzed by flow cytometry using a FACScan microfluorometer (BD Biosciences, San Jose, CA). A total of 10,000 events were counted for each condition.
sCD14 assay
Seminal fluid samples were a gift from A. Sunde (Trondheim, Norway). Normal semen was collected at the Department of Obstetrics and Gynecology of the Norwegian University of Science and Technology and processed as previously described (48). Cervical secretions were a gift from P. Crowley-Nowick (Brigham and Women’s Hospital, Boston, MA). Samples were collected using the Weck-cel collection method (49) during routine follow-up for abnormal Pap smears at Louisiana State Medical School (Shreveport, LA). Sponges were weighed before and after collection to correct for dilution. sCD14 was assayed by ELISA as described previously (50) (lower limit of detection, 0.78 ng/ml). All samples were taken after obtaining informed consent.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The immortalized End1/E6E7 epithelial cells and the primary
endocervical epithelial cells were evaluated for expression of mRNA for
the various TLRs by RT-PCR. HMEC cDNA and a bone marrow cDNA library
were used as controls. PCR primers and primer conditions are listed in
Table I
. While both End1/E6E7 and primary endocervical cells expressed
mRNA for TLR1, -2, -3, and -6, no PCR product was detected for TLR4 in
either cell population (Fig. 1). The
primary cells also failed to express message for TLR5. In contrast, a
TLR4 product of appropriate size was detected in HMEC endothelial cells
and the bone marrow library. The endocervical epithelial cells also
failed to express a significant amount of message for the
TLR4-associated molecule MD2 (Fig. 1). As expected, neither the
epithelial cell lines nor the HMEC cells expressed mRNA by RT-PCR for
the macrophage differentiation marker CD14 (data not shown).
Ectocervical (Ect1/E6E7) and vaginal (Vk2/E6E7) epithelial cells
demonstrated a pattern of TLR expression similar to that of the
endocervical epithelial cells (data not shown), suggesting that this
pattern of expression is consistent throughout the three compartments
of the lower female genital tract.
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A) and the primary
endocervical epithelial cells (Fig. 2B) expressed low levels
of TLR2 on the surface, but neither expressed TLR4. The specificity of
the Abs for TLR2 and TLR4 is demonstrated in Fig. 2C. Of
interest, we found that the HMEC endothelial cells expressed surface
TLR4 but failed to express TLR2 (data not shown), as has been
reported by others (51).
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B-mediated proinflammatory pathways
To determine whether N. gonorrhoeae infection of
epithelial cells could initiate a proinflammatory signal, cells were
assayed for a pretranscriptional event, the nuclear translocation of
the transcription factor NF-B. NF-B activation is central to the
inflammatory response to bacterial pathogens, leading to the release of
several proinflammatory cytokines and chemokines (reviewed in Ref.
52). We found evidence of NF-B activation by the EMSA
within 1 h of stimulation using both live N.
gonorrhoeae and bacterial lysates, suggesting that attachment and
invasion were not necessary for cellular activation (Fig. 3A). In fact, the bacterial
lysates appeared to be more potent than the live bacteria, perhaps
reflecting the availability of more inflammatory ligands in the
particulates compared with intact bacteria.
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B, IL-8 concentrations were
increased in culture supernatants in a time- and dose-dependent manner
when stimulated with bacterial lysates. Similar results were found when
supernatants were assayed for IL-6 (data not shown). sCD14 plays a role in host epithelial cell responses to N. gonorrhoeae in the lower female genital tract
Because the cells are grown in serum-free medium, they are not
exposed to the serum proteins sCD14 (54, 55, 56) and LBP
(57), which have been shown by many groups to enhance
responses to LPS. sCD14 has also been shown to interact with other
bacterial ligands, including mycobacterial lipoarabinomannan
(58) and mycobacterial cell wall components
(50), peptidoglycan (59, 60) and cell wall
components of Staphylococcus aureus (61), and
bacterial outer membrane lipoproteins (62). When the
culture medium was supplemented with sCD14, the endocervical epithelial
cells were more sensitive to activation by N. gonorrhoeae
lysates, with the cytokine response to stimulatory concentrations of
bacteria enhanced at all levels of sCD14 tested (Fig. 4). Furthermore, high levels of sCD14
(>100 ng/ml) enabled cellular responses to normally substimulatory
concentrations of bacteria (Fig. 4). Identical results were obtained
using live bacteria (data not shown). No effect of LBP, either by
itself or in combination with sCD14, was seen (data not shown).
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Various components of the bacterial cell wall are capable of activating the proinflammatory response. In the case of Gram-negative bacteria, LPS, or more specifically the lipid A core of LPS, is believed to be the molecule responsible for the biological toxicity (63). However, other components of the Gram-negative cell wall, such as proteins, lipoproteins, peptidoglycan, porins, and a variety of phospholipids, have been shown in other settings to activate of the inflammatory response. Furthermore, an LPS-deficient mutant of Neisseria meningitidis has been shown to be a potent inflammatory stimulus (64).
Because endocervical epithelial cells lacked the known components of
the LPS signaling complex, specifically TLR4 and MD2, we suspected that
they would be unresponsive to LPS. To test this, cells were incubated
with increasing amounts of LPS and other purified bacterial products
and assessed for IL-8 and IL-6 release. Considering the requirement of
sCD14 for LPS responses in cells lacking membrane CD14, medium was
supplemented with recombinant sCD14 (54, 55, 56). As
demonstrated in Fig. 6A, the
immortalized endocervical epithelial cells failed to increase IL-8
secretion in response to protein-free preparations of endotoxin derived
from N. gonorrhoeae and E. coli. They also failed
to translocate the transcription factor NF-B as assayed by the EMSA
upon LPS stimulation (data not shown). In contrast, they responded
quite well to other bacterial preparations, including synthetic
bacterial lipopeptides from T. pallidum (47-L) and M.
fermentans (sMALP-2), sPG, and HKLM. All these preparations have
been shown previously to be ligands for TLR2 (23, 24, 25, 26, 27, 29),
or TLR2 in concert with TLR6 (30). Likewise, primary
endocervical epithelial cells were activated by gonococcal lysates and
the TLR2 ligands 47-L and sMALP-2, but were unresponsive to the two LPS
preparations tested (Fig. 6B). Primary ectocervical
epithelial cells were also LPS unresponsive (data not shown).
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The C-terminal domain of MyD88 has been shown to act as a
dominant-negative inhibitor of NF-B activation by both IL-1
(65) and many of the TLRs (46). We
hypothesized that such a MyD88-DN mutant would block the N.
gonorrhoeae-induced NF-B activation that we observed in our
epithelial cells. To test this idea, we used an NF-B-dependent
ELAM-luciferase reporter. Cells were transiently cotransfected with the
reporter plasmid and either the expression plasmid containing the
dominant-negative MyD88 construct or a control vector. Cells were then
stimulated with increasing concentrations of the gonococcal lysates and
assayed for activation of the luciferase reporter. As shown in Fig. 7, overexpression of the
dominant-negative MyD88 blocked gonococcal-induced activation of the
luciferase reporter as well as that of IL-1, which uses the same
intracellular signals as the TLRs. In contrast to MyD88, we were unable
to see consistent inhibition of the crude lysates with either a TLR2
dominant-negative construct or TLR2-blocking Abs, suggesting that the
ligands in the gonococcal lysates responsible for the cellular
activation were using more than one TLR in the MyD88-dependent
activation (data not shown).
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We hypothesized that the absence of TLR4 and MD2 was responsible
for the LPS unresponsiveness by epithelial cells in the lower female
genital tract. To test this hypothesis, we transiently cotransfected
the endocervical epithelial cell line with the ELAM-luciferase reporter
plasmid and the expression plasmids containing the genes for human TLR4
and MD2. Cells were then stimulated with both N. gonorrhoeae
LOS and E. coli LPS, in the presence of sCD14 and assayed
for activation of the luciferase reporter. As predicted, we found that
the combination of TLR4 and MD2 conferred responsiveness to the two
different LPS preparations, while expression of TLR4 or MD2 alone did
not (Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results demonstrate that epithelial cells in the lower genital
tract have evolved a unique mechanism for the recognition of
Gram-negative pathogens. In contrast to professional phagocytic cells,
the epithelial cells of the mucosal surface lack the key components of
the LPS response machinery, TLR4 and MD2, and, consistent with this,
lack cytokine responsiveness to purified LPS preparations. Epithelial
cells in other mucosal sites have also been shown to have various
degrees of LPS responsiveness. Perhaps most confusing are the data on
gastrointestinal epithelial cells, where the ability of LPS to
up-regulate proinflammatory cytokines appears to vary with the cell
line tested (67, 68, 69, 70). In addition, airway epithelial cells
have demonstrated sensitivity to LPS by NF-B activation, cytokine
production, and up-regulation of various defense molecules
(71, 72, 73, 74), and LPS has been shown to simulated cytokine
expression by human gingival epithelial cells (75, 76, 77).
None of these studies identified host cell surface moieties linked to
epithelial-LPS interactions.
Although considerable effort has been aimed at the elucidation of TLR signaling in professional immune cells, the roles of various TLR-related molecules in mucosal epithelial cell recognition of bacterial components remain poorly understood, with gastrointestinal epithelial cells having been studied the most extensively. For example, colon cancer cell lines as well as intestinal epithelial cells derived from patients with idiopathic inflammatory bowel disease have been reported to express functional TLR4 (78). In contrast, epithelial cells in normal adult intestinal tissue appear to be TLR4 deficient by immunohistochemistry (79), and it has been reported that several intestinal epithelial cell lines lack TLR4 and MD2, accounting for their LPS hyporesponsiveness (80). In addition, Asai et al. (81) recently reported that gingival epithelial cells also fail to express TLR4 and respond to Gram-negative bacteria via TLR2 ligands.
Our findings are the first to characterize the expression of the known TLRs in normal, nontumorigenic epithelial cells and the first to examine the expression in the mucosal surface of the female genital tract. The similarity between our data and the recent reports on digestive tract epithelium by Abreu et al. (80) and Asai et al. (81) suggests that nonsterile mucosa has evolved a unique mechanism for bacterial recognition that differs from that used in sterile sites. The unique TLR expression pattern, in particular the lack of TLR4 and the associated molecule MD2, suggests that the cervicovaginal epithelium is capable of responding to Gram-negative pathogens in the absence of endotoxin recognition. This is in contrast to the mechanism used by phagocytes (15, 17, 82), dendritic cells (83), and endothelial cells (51, 84), which express TLR4 and are highly sensitive to LPS activation through the Toll/IL-1 signaling pathway.
We can only speculate about the possible evolutionary pressure that would lead to such differential expression of TLR4. It has been suggested that the lack of TLR4 expression in intestinal epithelial cells might prevent the constant proinflammatory gene activation that could occur with exposure to normal enteric flora and might account for some of the chronic intestinal inflammation associated with disorders such as inflammatory bowel disease (78, 79, 80). The lower female genital tract also has a complex ecosystem, and it is possible that a similar model might exist in this compartment as well. Certainly, a threshold of sensitivity to bacterial components in the genital tract is required to avoid unnecessary inflammation. While inflammation is essential for clearing bacterial infections, excessive inflammation is particularly detrimental to the defense function of the mucosal surface. For example, it has been shown that inflammation increases the risk of sexual transmission of pathogens such as HIV-1 (7).
Our results also suggest that sCD14 has an LPS/TLR4-independent role in the genital tract epithelial cell responses to Gram-negative bacteria, enhancing the proinflammatory cytokine response to live bacteria and bacterial lysates. Even low concentrations of sCD14 (1–10 ng/ml) that would be below the detection limit of our assay because of sample dilution have effects on the cytokine response when the inoculum of bacteria is high. Furthermore, high concentrations of sCD14, as might be found during menses or following intercourse, may act to fortify the host defense under conditions of increased risk of infection by augmenting the inflammatory cytokine response. The precise molecular mechanism by which sCD14 enhances TLR-mediated cellular responses to bacterial membranes is unclear. Others have shown that membrane CD14 can facilitate signaling via TLR2 ligands including lipoproteins (62, 85) and peptidoglycan (23, 24). An enhancing effect of sCD14 was also found by Asai and colleagues regarding Porphyromonas gingivalis fimbrial lipoproteins and TLR2 (81). Our data go further to suggest that sCD14 is capable of enhancing the proinflammatory response not only to purified ligand, but also to bacterial lysates and live bacteria. This could be accomplished either by directly presenting ligand to a TLR or by increasing the availability of ligands by interactions with the bacterial membrane.
Thus, while TLR4 may be essential for the response to Gram-negative pathogens during bacteremia and sepsis, other TLRs may be more important for the recognition of Gram-negative bacteria at specific sites, such as the mucosal surface of the female genital tract. A better understanding of the regulation of TLR expression and its influence on bacterial recognition may provide insight into the pathogenesis of a variety of infectious diseases, in particular the pathophysiology of sexually transmitted diseases and the deleterious sequelae associated with chronic genital tract inflammation.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robin R. Ingalls, Department of Infectious Diseases, Boston Medical Center, Boston University School of Medicine, 650 Albany Street, Boston, MA 02118. E-mail address: ringalls{at}bu.edu
3 Abbreviations used in this paper: TLR, Toll-like receptor; HKLM, heat-killed Listeria monocytogenes; LBP, LPS binding protein; LOS, lipooligosaccharide; sCD14, soluble CD14; sMALP-2, synthetic lipopeptide based on the full-length MALP-2; sPG, soluble peptidoglycan.
Received for publication October 22, 2001. Accepted for publication January 9, 2002.
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C. M. O'Connell, R. R. Ingalls, C. W. Andrews Jr., A. M. Scurlock, and T. Darville Plasmid-Deficient Chlamydia muridarum Fail to Induce Immune Pathology and Protect against Oviduct Disease J. Immunol., September 15, 2007; 179(6): 4027 - 4034. [Abstract] [Full Text] [PDF]
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 K. Nasu, H. Itoh, A. Yuge, M. Nishida, and H. Narahara Human oviductal epithelial cells express Toll-like receptor 3 and respond to double-stranded RNA: Fallopian tube-specific mucosal immunity against viral infection Hum. Reprod., February 1, 2007; 22(2): 356 - 361. [Abstract] [Full Text] [PDF]
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J. P. Mackern-Oberti, M. Maccioni, C. Cuffini, G. Gatti, and V. E. Rivero Susceptibility of Prostate Epithelial Cells to Chlamydia muridarum Infection and Their Role in Innate Immunity by Recruitment of Intracellular Toll-Like Receptors 4 and 2 and MyD88 to the Inclusion Infect. Immun., December 1, 2006; 74(12): 6973 - 6981. [Abstract] [Full Text] [PDF]
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A. Riccioli, D. Starace, R. Galli, A. Fuso, S. Scarpa, F. Palombi, P. De Cesaris, E. Ziparo, and A. Filippini Sertoli Cells Initiate Testicular Innate Immune Responses through TLR Activation J. Immunol., November 15, 2006; 177(10): 7122 - 7130. [Abstract] [Full Text] [PDF]
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 S. Trapp, S. G. Turville, and M. Robbiani Slamming the door on unwanted guests: why preemptive strikes at the mucosa may be the best strategy against HIV J. Leukoc. Biol., November 1, 2006; 80(5): 1076 - 1083. [Full Text] [PDF]
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J. Heidemann, W. Domschke, T. Kucharzik, and C. Maaser Intestinal microvascular endothelium and innate immunity in inflammatory bowel disease: a second line of defense? Infect. Immun., October 1, 2006; 74(10): 5425 - 5432. [Full Text] [PDF]
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R. N. Fichorova, R. T. Trifonova, R. O. Gilbert, C. E. Costello, G. R. Hayes, J. J. Lucas, and B. N. Singh Trichomonas vaginalis Lipophosphoglycan Triggers a Selective Upregulation of Cytokines by Human Female Reproductive Tract Epithelial Cells. Infect. Immun., October 1, 2006; 74(10): 5773 - 5779. [Abstract] [Full Text] [PDF]
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 S. Santos-Sierra, D. T. Golenbock, and P. Henneke Toll-like receptor-dependent discrimination of streptococci Innate Immunity, October 1, 2006; 12(5): 307 - 312. [Abstract] [PDF]
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O. Equils, D. Lu, M. Gatter, S. S. Witkin, C. Bertolotto, M. Arditi, J. A. McGregor, C. F. Simmons, and C. J. Hobel Chlamydia Heat Shock Protein 60 Induces Trophoblast Apoptosis through TLR4 J. Immunol., July 15, 2006; 177(2): 1257 - 1263. [Abstract] [Full Text] [PDF]
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 G. Soboll, L. Shen, and C. R. Wira Expression of Toll-Like Receptors (TLR) and Responsiveness to TLR Agonists by Polarized Mouse Uterine Epithelial Cells in Culture Biol Reprod, July 1, 2006; 75(1): 131 - 139. [Abstract] [Full Text] [PDF]
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J. M. Andersen, D. Al-Khairy, and R. R. Ingalls Innate Immunity at the Mucosal Surface: Role of Toll-Like Receptor 3 and Toll-Like Receptor 9 in Cervical Epithelial Cell Responses to Microbial Pathogens Biol Reprod, May 1, 2006; 74(5): 824 - 831. [Abstract] [Full Text] [PDF]
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 C. M. O'Connell, I. A. Ionova, A. J. Quayle, A. Visintin, and R. R. Ingalls Localization of TLR2 and MyD88 to Chlamydia trachomatis Inclusions: EVIDENCE FOR SIGNALING BY INTRACELLULAR TLR2 DURING INFECTION WITH AN OBLIGATE INTRACELLULAR PATHOGEN J. Biol. Chem., January 20, 2006; 281(3): 1652 - 1659. [Abstract] [Full Text] [PDF]
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W. A. Derbigny, M. S. Kerr, and R. M. Johnson Pattern Recognition Molecules Activated by Chlamydia muridarum Infection of Cloned Murine Oviduct Epithelial Cell Lines J. Immunol., November 1, 2005; 175(9): 6065 - 6075. [Abstract] [Full Text] [PDF]
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J. Zhang, G. Li, A. Bafica, M. Pantelic, P. Zhang, H. Broxmeyer, Y. Liu, L. Wetzler, J. J. He, and T. Chen Neisseria gonorrhoeae Enhances Infection of Dendritic Cells by HIV Type 1 J. Immunol., June 15, 2005; 174(12): 7995 - 8002. [Abstract] [Full Text] [PDF]
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A. Fazeli, C. Bruce, and D.O. Anumba Characterization of Toll-like receptors in the female reproductive tract in humans Hum. Reprod., May 1, 2005; 20(5): 1372 - 1378. [Abstract] [Full Text] [PDF]
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M. L. Peterson, K. Ault, M. J. Kremer, A. J. Klingelhutz, C. C. Davis, C. A. Squier, and P. M. Schlievert The Innate Immune System Is Activated by Stimulation of Vaginal Epithelial Cells with Staphylococcus aureus and Toxic Shock Syndrome Toxin 1 Infect. Immun., April 1, 2005; 73(4): 2164 - 2174. [Abstract] [Full Text] [PDF]
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K. S. Grant-Tschudy and C. R. Wira Hepatocyte Growth Factor Regulation of Uterine Epithelial Cell Transepithelial Resistance and Tumor Necrosis Factor {alpha} Release in Culture Biol Reprod, April 1, 2005; 72(4): 814 - 821. [Abstract] [Full Text] [PDF]
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T. M. Schaefer, J. V. Fahey, J. A. Wright, and C. R. Wira Innate Immunity in the Human Female Reproductive Tract: Antiviral Response of Uterine Epithelial Cells to the TLR3 Agonist Poly(I:C) J. Immunol., January 15, 2005; 174(2): 992 - 1002. [Abstract] [Full Text] [PDF]
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R. N. Fichorova, F. Zhou, V. Ratnam, V. Atanassova, S. Jiang, N. Strick, and A. R. Neurath Anti-Human Immunodeficiency Virus Type 1 Microbicide Cellulose Acetate 1,2-Benzenedicarboxylate in a Human In Vitro Model of Vaginal Inflammation Antimicrob. Agents Chemother., January 1, 2005; 49(1): 323 - 335. [Abstract] [Full Text] [PDF]
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 T. Hirata, Y. Osuga, Y. Hirota, K. Koga, O. Yoshino, M. Harada, C. Morimoto, T. Yano, O. Nishii, O. Tsutsumi, et al. Evidence for the Presence of Toll-Like Receptor 4 System in the Human Endometrium J. Clin. Endocrinol. Metab., January 1, 2005; 90(1): 548 - 556. [Abstract] [Full Text] [PDF]
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P. A. Pioli, E. Amiel, T. M. Schaefer, J. E. Connolly, C. R. Wira, and P. M. Guyre Differential Expression of Toll-Like Receptors 2 and 4 in Tissues of the Human Female Reproductive Tract Infect. Immun., October 1, 2004; 72(10): 5799 - 5806. [Abstract] [Full Text] [PDF]
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R.N. Fichorova, M. Bajpai, N. Chandra, J.G. Hsiu, M. Spangler, V. Ratnam, and G.F. Doncel Interleukin (IL)-1, IL-6, and IL-8 Predict Mucosal Toxicity of Vaginal Microbicidal Contraceptives Biol Reprod, September 1, 2004; 71(3): 761 - 769. [Abstract] [Full Text] [PDF]
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 A. Muir, G. Soong, S. Sokol, B. Reddy, M. I. Gomez, A. van Heeckeren, and A. Prince Toll-Like Receptors in Normal and Cystic Fibrosis Airway Epithelial Cells Am. J. Respir. Cell Mol. Biol., June 1, 2004; 30(6): 777 - 783. [Abstract] [Full Text] [PDF]
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T. Guthrie, S. Y. C. Wong, B. Liang, L. Hyland, S. Hou, E. A. Hoiby, and S. R. Andersen Local and Systemic Antibody Responses in Mice Immunized Intranasally with Native and Detergent-Extracted Outer Membrane Vesicles from Neisseria meningitidis Infect. Immun., May 1, 2004; 72(5): 2528 - 2537. [Abstract] [Full Text] [PDF]
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K. S. Kobayashi and R. A. Flavell Shielding the double-edged sword: negative regulation of the innate immune system J. Leukoc. Biol., March 1, 2004; 75(3): 428 - 433. [Abstract] [Full Text] [PDF]
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T. Darville, J. M. O'Neill, C. W. Andrews Jr., U. M. Nagarajan, L. Stahl, and D. M. Ojcius Toll-Like Receptor-2, but Not Toll-Like Receptor-4, Is Essential for Development of Oviduct Pathology in Chlamydial Genital Tract Infection J. Immunol., December 1, 2003; 171(11): 6187 - 6197. [Abstract] [Full Text] [PDF]
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P. L. Fisette, S. Ram, J. M. Andersen, W. Guo, and R. R. Ingalls The Lip Lipoprotein from Neisseria gonorrhoeae Stimulates Cytokine Release and NF-{kappa}B Activation in Epithelial Cells in a Toll-like Receptor 2-dependent Manner J. Biol. Chem., November 21, 2003; 278(47): 46252 - 46260. [Abstract] [Full Text] [PDF]
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Y. Asai, T. Jinno, and T. Ogawa Oral Treponemes and Their Outer Membrane Extracts Activate Human Gingival Epithelial Cells through Toll-Like Receptor 2 Infect. Immun., February 1, 2003; 71(2): 717 - 725. [Abstract] [Full Text] [PDF]
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K. Maisey, G. Nardocci, M. Imarai, H. Cardenas, M. Rios, H. B. Croxatto, J. E. Heckels, M. Christodoulides, and L. A. Velasquez Expression of Proinflammatory Cytokines and Receptors by Human Fallopian Tubes in Organ Culture following Challenge with Neisseria gonorrhoeae Infect. Immun., January 1, 2003; 71(1): 527 - 532. [Abstract] [Full Text] [PDF]
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