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,¶,
Departments of
* Immunology,
Surgery, and
Pediatrics, Mayo Clinic, Rochester, MN 55905;
Department of Surgery, Nippon Medical School, Bunkyoku, Tokyo; and
¶ Tsukuba Memorial Hospital, Tsukuba, Ibaraki, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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While many types of cells may participate in vertebrate innate immune responses, adaptive immune responses are triggered predominantly by dendritic cells (DC) (11). Upon activation of TLR, DC briefly increase phagocytosis, migrate to lymph nodes (12), process and present phagocytosed Ags, and undergo a phenotypic maturation resulting in high expression of adhesive and costimulatory proteins, all of which help to activate naive T lymphocytes (13). Maturation of DC, and the subsequent activation of the adaptive immune system, must also occur in the absence of exogenous stimuli (14, 15), because T lymphocyte responses are vital to the defense against noncytopathic viruses, tumors, and transplants (16). Endogenous molecules generated at the very inception of tissue disease that are capable of activating TLR on DC, and thus leading to their maturation in the absence of exogenous stimuli, have not been identified.
Because injured or infected tissues in plants and invertebrates generate fragments of endogenous molecules that activate immunity through sentinel receptors, we questioned whether vertebrates use a similar molecular pathway to monitor tissue disease. In support of this, we recently found that fragments of heparan sulfate, an acidic polysaccharide ordinarily found in cell membranes and extracellular matrices (17), activates DC (18). Heparan sulfate is rapidly shed from cell surfaces and basement membranes as a result of tissue injury (19, 20), in the course of general inflammation (21, 22), and is also shed as a result of tumor cell migration and metastasis (23). In this work we report that soluble heparan sulfate uses TLR4 in the activation of DC, thus suggesting that vertebrate TLR monitor perturbations to the well-being of tissues. Therefore, plants, invertebrates, and highly evolved vertebrates maintain receptor systems that are capable of recognizing general signals of tissue disease manifest by fragmentation of endogenous molecules.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ultrapure heparan sulfate (Super Special Grade) and chondroitin sulfate B were obtained from Seikagaku (Falmouth, MA), and LPS derived from Escherichia coli. was obtained from Sigma-Aldrich (St. Louis, MO). CpG DNA, non-CpG DNA, and inhibitory CpG DNA were synthesized and phosphorothioate modified. The following FITC-conjugated mAbs were obtained from BD PharMingen (San Diego, CA): HM40-3 (anti-CD40), 16-10A1 (anti-CD80: B7-1), GL1 (anti-CD86: B7-2), G235-2356 (hamster IgG isotype standard), R35-95 (rat IgG isotype standard). The following biotin-conjugated Abs were obtained from BD PharMingen: AF6-88.5 (anti-H-2Kb) and AF6-120.1 (anti-I-Ab). PE-conjugated streptavidin and unconjugated 2.4G2 (anti-CD16/CD32) were also purchased from BD PharMingen. CD4 MicroBeads were purchased from Miltenyi Biotec (Auburn, CA). LPS removal resin (END-X B15) was from Associates of Cape Cod (Falmouth, MA). Recombinant mouse GM-CSF was purchased from R&D Systems (Minneapolis, MN). Rhodobacter sphaeroides diphosphoryl lipid A (Rs-DPLA) was a gift from N. Quereshi (University of Wisconsin, Madison, WI). Synthetic single-stranded oligonucleotides were dissolved in TE (10 mM Tris, 1 mM EDTA), purified by gel filtration chromatography, and quantitated spectrophotometrically.
Single-stranded oligonucleotides
The following sequences were used for inhibition or stimulation of cells: CpG sequence (ODN1826) 5'-TCCATGACGTTCCTGACGTT-3' (24); non-CpG sequence (ODN1911) 5'-TCCAGGACTTTCCTCAGGTT-3' (24); and CpG inhibitory sequence 5'-TCCATGGCGGGCCTGGCGGG-3' (65).
Cell isolation and culture
DC were generated from murine bone marrow culture as previously described (18). Briefly, bone marrow was flushed from the long bones of C57BL10ScNCr (National Cancer Institute, Bethesda, MD), C57BL6J, BALBcJ, C57BL10SnJ, C3H/HeJ, or C3H/HeSnJ mice (The Jackson Laboratory, Bar Harbor, ME) and depleted of red cells with ammonium chloride. At day 3 of culture, floating cells were gently removed and fresh medium containing 3.3 ng/ml GM-CSF was added. At day 6 or 7 of culture, nonadherent cells and loosely adherent proliferating cell aggregates were harvested for analysis or stimulation. RAW 264.7 (ATCC no. TIB71; American Type Culture Collection, Manassas, VA) were maintained in culture as recommended by the supplier. Splenocytes from 8-wk-old BALB/cJ mice (H-2d) were purified by density centrifugation (Ficoll Paque Plus (Amersham Pharmacia Biotech, Uppsala, Sweden)), washed with PBS, and resuspended in RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Gaithersburg, MD), and incubated at 37°C, 5% CO2.
Stimulation of DC and RAW 264.7
Cells were stimulated with 10 µg/ml chondroitin sulfate, 150 ng/ml CpG DNA, 10 ng/ml LPS, and 10 µg/ml heparan sulfate or PBS in culture medium for 24 h, unless otherwise indicated. Rs-DPLA, or CpG inhibitory DNA, were added to cell culture as indicated 1 h before stimulation. In some experiments, stimulants were pretreated by 24-h incubation on a nutator with endotoxin removal resin, containing Limulus anti-LPS factor (LALF) covalently linked to silica beads, or were boiled for 10 min at 100°C.
Flow cytometric analysis
Flow cytometric analysis was performed as described by Kodaira et al. (25). Cells were incubated with anti-CD16/CD32 Ab, then stained with the indicated Abs and analyzed by FACScan using CellQuest software (BD Biosciences, San Jose, CA).
Phase microscopy
Phase microscopy images of cells in active culture were captured at x100 on an inverted microscope.
Modified MLRs
Immature C3H/HeJ, C3H/HeSnJ (H-2k), or BALB/cJ (H-2d) DC were stimulated, then washed and resuspended at various concentrations in 100 µl/well of medium in 96-well round-bottom plates. Splenocytes from BALB/cJ mice (H-2d) were added at various concentrations to DC at 100 µl/well and incubated at 37°C, 5% CO2. After 3, 4, 5, and 6 days of coculture, 2 mCi/ml [3H]thymidine (ICN Pharmaceuticals, Irvine, CA) was added for 16 h. [3H]Thymidine incorporation into DNA was analyzed using a liquid scintillation counter (Wallac, Turku, Finland). Student’s t test was used for statistical analysis. In separate experiments, immature female C57BL/10SnJ and C57BL10/ScNCr DC were stimulated in the presence of multiple soluble Ags from bovine serum for 24 h, then washed three times and irradiated with 15 Gy. CD4+ cells were isolated from combined spleens and axillary lymph nodes of female C3H/HeSnJ by staining with CD4 MicroBeads and positively selected on a LS+ column using a VarioMACS instrument (Miltenyi Biotec). DC were cocultured at various concentrations with CD4+ Th cells at various concentrations, and thymidine incorporation was measured after 3, 4, 5, and 6 days as previously described.
NF-
B translocation analysis
Nuclear translocation of NF-B was analyzed using an EMSA. DC
or RAW 264.7 cells were stimulated with the indicated reagents and for
the indicated times, then washed three times with ice-cold PBS. Cells
were lysed by scraping and nuclear protein was extracted from washed
nuclei as previously described (26).
EMSA was performed according to a modification of a previously
described procedure (27). Nuclear extract (10 µg) was
preincubated on ice in poly(dI-dC)-containing binding buffer (see
below). Where indicated, cold competitive oligonucleotides were
included during the preincubation period.
32P-labeled oligonucleotide probe containing two
tandem NF-B binding sites (28) was added (2.5 x
104 cpm or 
2.5 fmol/reaction), and the
reactions were incubated for 15 min at 25°C. The final binding
reactions (20 µl) contained 12.5 mM HEPES, 87.5 mM NaCl, 1 mM DTT,
0.15 mM EDTA, 0.02% Nonidet P-40, 12.5% glycerol, and 100 µg/ml
poly(dI-dC). The samples were electrophoresed through 4%
polyacrylamide gels (25:1 acrylamide:bisacrylamide) in 45 mM
Tris-borate buffer containing 1 mM EDTA (pH 8). Gels were dried under
vacuum and radiolabeled species were detected by capture on a Kodak
storage phosphor screen (Kodak, Rochester, NY) and revealed by a
Bio-Rad molecular imager FX (Bio-Rad, Irvine, CA). Visualization and
quantitative analysis were done using Bio-Rad Quantity One
software.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To explore the potential involvement of TLR in the maturation of
DC, we used a model system in which DC are driven to mature by
established agonists of TLR (29, 30). Fig. 1
shows that immature DC, grown from
mouse bone marrow culture with GM-CSF for 6–7 days, are induced to
mature by soluble heparan sulfate, by LPS, the active agent of
bacterial endotoxin (31), and by single-stranded,
nonmethylated DNA with a bacterial CpG motif (CpG DNA). Maturation of
DC is manifest by a progressive increase in expression of cell surface
molecules that aid in T lymphocyte stimulation—CD80 (B7-1), CD86
(B7-2), CD40, and MHC class II (Fig. 1 and data not shown)—consistent
with findings established in previous reports (18, 29, 30). Limiting concentrations of heparan sulfate, LPS and CpG DNA
induce maturation at the same rate and to the same extent (Fig. 1).
Heparan sulfate, LPS, and CpG DNA (Fig. 1D) induced the
formation of cell aggregates, which are commonly seen as DC mature in
culture (32). These results show that heparan sulfate,
LPS, and CpG DNA induce similar maturation of DC.
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and data no
shown). Although heparan sulfate used in these experiments was
chromatography purified and deemed ultrapure by amino acid autoanalyzer
and cellulose acetate strip electrophoresis, the absence of LPS was
confirmed by the following experiments. First, adsorption of heparan
sulfate with a LPS-binding protein, LALF, linked to silica beads that
were then removed by centrifugation did not reduce, and in fact
enhanced, the ability of heparan sulfate to induce DC maturation (Fig. 2). However, the same treatment of LPS
eliminated its ability to induce DC maturation (18, 35 and
Fig. 2). Heparan sulfate has little or no reactivity with LALF
(36). Second, digestion of heparan sulfate by deaminative
cleavage eliminated the stimulatory capacity of heparan sulfate but had
no effect on DC maturation induced by LPS (18). Third, the
inhibition and signaling kinetics of heparan sulfate differed
markedly from that of LPS (see below). The absence of contaminating
proteins was confirmed by the following experiment. Boiling of heparan
sulfate, which inactivates the ability of proteins to stimulate TLR4
(6, 7, 8, 37), had no effect on DC maturation induced by
heparan sulfate (Ref. 18 and data not shown).
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Because DC appear to respond to heparan sulfate in the same way as
they do to LPS and CpG DNA, we asked whether heparan sulfate might use
the same cellular receptors as these agonists. To address this
question, we tested whether inhibitors of the receptors used by LPS and
CpG DNA inhibit activation of DC by heparan sulfate. Rs-DPLA is a
competitive inhibitor of LPS and other TLR4 agonists (5, 38, 39). As Fig. 3A shows, DC incubated with Rs-DPLA did
not mature in response to heparan sulfate. In contrast, DC incubated
with a competitive inhibitor of CpG DNA4 did
mature in the presence of heparan sulfate (Fig. 3B). Taken together, these
results suggest that the maturation of DC induced by heparan sulfate
may require TLR4, but not TLR9, the receptor that recognizes CpG DNA
(40).
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Because Rs-DPLA inhibits the action of multiple extracellular proteins
that may specifically aid in LPS signaling through TLR4, one might
expect Rs-DPLA to inhibit other TLR4 agonists at different doses. We
found that Rs-DPLA is a more potent inhibitor of DC maturation induced
by heparan sulfate, as compared with LPS (requiring a 6- to 7-fold
lower dose of Rs-DPLA), despite the fact that we used limiting
concentrations of heparan sulfate and LPS (Fig. 3A). Rs-DPLA
inhibition was not due to interaction with MyD88 or other intracellular
signaling molecules that are shared by TLR (40), because
Rs-DPLA did not inhibit CpG DNA-induced maturation (Fig. 3A). These data are consistent with a model in which DC
activation by heparan sulfate and DC activation by LPS both depend on
TLR4 but differ with regard to the types of interactions with TLR4 and
coreceptors.
Effect of TLR4 mutation on DC maturation induced by heparan sulfate
To determine whether DC maturation induced by heparan sulfate
requires functionally active TLR4, we tested DC cultured from C3H/HeJ
mice. C3H/HeJ mice have a single amino acid mutation
(Pro712 to His712) in the
conserved cytoplasmic Toll-IL-1R domain of TLR4 and do not respond to
the presence of LPS (31). The mutation in the
TLR4 gene of C3H/HeJ mice abrogates the interaction
of TLR4 with MyD88, a cytoplasmic adaptor molecule shared by TLR and
required for full responses (31, 44). Fig. 4A shows that DC from C3H/HeJ
mice do not mature in response to either heparan sulfate or LPS,
whereas DC from TLR4 wild-type mice with the same genetic background
mature normally. In contrast, C3H/HeJ DC do mature in response to CpG
DNA (Fig. 4A), showing that these mutant DC are capable of
responding to stimulation via TLR9.
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Although the results given above suggest that functional TLR4 is
required for DC to respond to heparan sulfate, the results do not
exclude the possibility that C3H/HeJ mice may have another defect, such
as a deficient production of IFN-
(45), that may impair
responses to heparan sulfate. To test whether a defect in TLR4, and not
some other defect, abrogates maturation of DC induced by heparan
sulfate, we tested DC from C57BL/10ScNCr mice. C57BL/10ScNCr and
C57BL/10ScCr mice have a deletion in chromosome 4 that encompasses the
TLR4 gene and, like C3H/HeJ mice, do not respond to the
presence of LPS (31). C57BL/10ScCr have been used
extensively to study the function of TLR4; however, C57BL/10ScCr mice
have an additional mutation in their IL-12R
2 gene that is
not found in C57BL/10ScNCr mice (46). Fig. 4B
shows that DC from C57BL/10ScNCr mice do not mature in response to
either heparan sulfate or LPS, whereas DC from C57BL/10SnJ mice, a TLR4
wild-type strain with the same genetic background, mature normally. It
should be noted that DC from C57BL/10SnJ mice appear to express higher
basal levels of CD80, CD86, and CD40 than DC from C57BL/10ScNCr mice,
but this does not effect the clear difference between the strains in
response to stimulation with heparan sulfate or LPS. DC from
C57BL/10SnJ mice also increase expression of MHC class II
(I-Ab) and MHC class I
(H-2Kb) in response to heparan sulfate, whereas
DC from C57BL/10ScNCr mice do not (data not shown). Like DC
from C3H/HeJ mice, DC from C57BL/10ScNCr mice do mature in response to
CpG DNA (Fig. 4B), indicating that DC from both strains of
TLR4 mutant mice are capable of responding to stimulation via
TLR9.
Effect of TLR4 mutation on activation of T lymphocytes by DC stimulated with heparan sulfate
We tested whether TLR4 is essential for the increase in T
lymphocyte stimulation by DC induced to mature by heparan sulfate
(18). To address this we used a modified MLR in which
stimulated DC are mixed in culture with splenocytes from a different
strain of mice. The cellular proliferation that ensues is measured as
an indicator of T lymphocyte stimulation and acts as an in vitro model
of activation of acquired immune responses that may lead to allogeneic
transplant rejection. Fig. 5A
shows that DC cultured from C3H/HeJ mice do not increase their
stimulatory capacity in response to either heparan sulfate or LPS,
whereas DC from TLR4 wild-type mice increase their stimulatory capacity
significantly in response to both heparan sulfate and LPS.
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B shows that DC expressing
wild-type TLR4 stimulated with heparan sulfate and exposed to Ag caused
T cell proliferation, whereas DC lacking TLR4 did not. These results
show that TLR4 on DC is required for heparan sulfate to cause
functional DC maturation.
Heparan sulfate induced nuclear translocation of NF-B
Maturation of DC by TLR activation appears to require nuclear
translocation of NF-B, which is an intracellular mediator of TLR
signals (3, 48). We tested whether stimulation of DC with
heparan sulfate induces rapid increases in nuclear NF-B, which is
capable of binding to a DNA promoter sequence, as measured by gel EMSA.
Fig. 6A shows strong increases
in NF-B in DC stimulated by heparan sulfate, LPS, and CpG DNA.
Because unstimulated DC contain basal levels of NF-B, whereas
unstimulated macrophages contain negligible levels of NF-B (Fig. 6B), we used macrophages, in which nuclear translocation of
NF-B occurs in response to LPS and heparan sulfate
(49), to address this question in further detail. We used
the murine macrophage cell line RAW 264.7, which responds to LPS in a
TLR4-dependent manner (50). Fig. 6C shows that
heparan sulfate and LPS induce NF-B translocation in a
dose-dependent manner. Fig. 6D shows that the kinetics of
NF-B translocation induced by heparan sulfate is significantly
slower than those induced by LPS. These data suggest that, although
heparan sulfate and LPS give rise to similar functional outcomes, the
intracellular signals they generate differ, perhaps because of
differences in extracellular molecular interactions with TLR4 and
coreceptors.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nearly every type of tissue injury, infection, and inflammation leads to shedding of heparan sulfate from mammalian cells (19, 20, 21, 22, 56). Activation of complement (21, 54, 57), neutrophils (58, 59, 60), or platelets (22), as well as the acidic environment of tissue damage (22), leads to the generation of soluble heparan sulfate proteoglycan and its fragmentation. Soluble heparan sulfate is not found in healthy tissues in significant quantities, whereas the concentration of soluble saccharide in the tissue fluid of wounds (61), the synovium of arthritic joints (62), and the urine of infected individuals (63) is within the range we observe to stimulate DC. Similar concentrations of homogalacturonan fragments are needed to stimulate host defense in plants (52, 53).
Activation of TLR by endogenous molecules such as heparan sulfate may also play a role in causing disease. Administration of high-dose CpG DNA or LPS leads to a syndrome that mimics septic shock, resulting in multiorgan failure and death (31, 40). However, humans who have survived severe injuries, as may result from burns or surgery, can also succumb to a "septic" syndrome, dying from multiorgan failure without any evidence of bacterial or fungal infection (64).
While we report studies using an in vitro model system, we believe it will be important to determine that activation of TLR4 by heparan sulfate also occurs in vivo. Our preliminary studies in several model systems are consistent with this likelihood.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jeffrey L. Platt, Transplantation Biology, Mayo Clinic, 2-66 Medical Sciences Building, Rochester, MN 55905. E-mail address: platt.jeffrey{at}mayo.edu
3 Abbreviations used in this paper: TLR, Toll-like receptor; LBP, LPS-binding protein; LALF, Limulus anti-LPS factor; Rs-DPLA, Rhodobacter sphaeroides diphosphoryl lipid A; DC, dendritic cell.
Received for publication October 30, 2001. Accepted for publication March 12, 2002.
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References |
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A. Tsung, J. R. Klune, X. Zhang, G. Jeyabalan, Z. Cao, X. Peng, D. B. Stolz, D. A. Geller, M. R. Rosengart, and T. R. Billiar HMGB1 release induced by liver ischemia involves Toll-like receptor 4 dependent reactive oxygen species production and calcium-mediated signaling J. Exp. Med., November 26, 2007; 204(12): 2913 - 2923. [Abstract] [Full Text] [PDF]
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R. M. Levy, K. P. Mollen, J. M. Prince, D. J. Kaczorowski, R. Vallabhaneni, S. Liu, K. J. Tracey, M. T. Lotze, D. J. Hackam, M. P. Fink, et al. Systemic inflammation and remote organ injury following trauma require HMGB1 Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2007; 293(4): R1538 - R1544. [Abstract] [Full Text] [PDF]
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 Y. Peng, G. Han, H. Shao, Y. Wang, H. J. Kaplan, and D. Sun Characterization of IL-17+ Interphotoreceptor Retinoid-Binding Protein-Specific T Cells in Experimental Autoimmune Uveitis Invest. Ophthalmol. Vis. Sci., September 1, 2007; 48(9): 4153 - 4161. [Abstract] [Full Text] [PDF]
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S. P. Gondokaryono, H. Ushio, F. Niyonsaba, M. Hara, H. Takenaka, S. T. M. Jayawardana, S. Ikeda, K. Okumura, and H. Ogawa The extra domain A of fibronectin stimulates murine mast cells via Toll-like receptor 4 J. Leukoc. Biol., September 1, 2007; 82(3): 657 - 665. [Abstract] [Full Text] [PDF]
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A. H. Tang, G. J. Brunn, M. Cascalho, and J. L. Platt Pivotal Advance: Endogenous pathway to SIRS, sepsis, and related conditions J. Leukoc. Biol., August 1, 2007; 82(2): 282 - 285. [Abstract] [Full Text] [PDF]
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H. F. Rosenberg Toll-like receptors, endogenous ligands, and constitutive control (or, why I'm still standing at the podium): an interview with Dr. Jeffrey L. Platt J. Leukoc. Biol., August 1, 2007; 82(2): 286 - 287. [Full Text] [PDF]
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U. Koedel, U. M. Merbt, C. Schmidt, B. Angele, B. Popp, H. Wagner, H.-W. Pfister, and C. J. Kirschning Acute Brain Injury Triggers MyD88-Dependent, TLR2/4-Independent Inflammatory Responses Am. J. Pathol., July 1, 2007; 171(1): 200 - 213. [Abstract] [Full Text] [PDF]
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D. F. LaRosa, A. H. Rahman, and L. A. Turka The Innate Immune System in Allograft Rejection and Tolerance J. Immunol., June 15, 2007; 178(12): 7503 - 7509. [Abstract] [Full Text] [PDF]
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J. Fan, Y. Li, R. M. Levy, J. J. Fan, D. J. Hackam, Y. Vodovotz, H. Yang, K. J. Tracey, T. R. Billiar, and M. A. Wilson Hemorrhagic Shock Induces NAD(P)H Oxidase Activation in Neutrophils: Role of HMGB1-TLR4 Signaling J. Immunol., May 15, 2007; 178(10): 6573 - 6580. [Abstract] [Full Text] [PDF]
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 M.-F. Tsan and Baochong Gao Review: Pathogen-associated molecular pattern contamination as putative endogenous ligands of Toll-like receptors Innate Immunity, February 1, 2007; 13(1): 6 - 14. [Abstract] [PDF]
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D. P. Beiting, L. F. Gagliardo, M. Hesse, S. K. Bliss, D. Meskill, and J. A. Appleton Coordinated Control of Immunity to Muscle Stage Trichinella spiralis by IL-10, Regulatory T Cells, and TGF-beta J. Immunol., January 15, 2007; 178(2): 1039 - 1047. [Abstract] [Full Text] [PDF]
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 M. Jayachandran, G. J. Brunn, K. Karnicki, R. S. Miller, W. G. Owen, and V. M. Miller In vivo effects of lipopolysaccharide and TLR4 on platelet production and activity: implications for thrombotic risk J Appl Physiol, January 1, 2007; 102(1): 429 - 433. [Abstract] [Full Text] [PDF]
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A. Tsung, N. Zheng, G. Jeyabalan, K. Izuishi, J. R. Klune, D. A. Geller, M. T. Lotze, L. Lu, and T. R. Billiar Increasing numbers of hepatic dendritic cells promote HMGB1-mediated ischemia-reperfusion injury J. Leukoc. Biol., January 1, 2007; 81(1): 119 - 128. [Abstract] [Full Text] [PDF]
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 A. Shimamoto, T. H. Pohlman, S. Shomura, T. Tarukawa, M. Takao, and H. Shimpo Toll-Like Receptor 4 Mediates Lung Ischemia-Reperfusion Injury Ann. Thorac. Surg., December 1, 2006; 82(6): 2017 - 2023. [Abstract] [Full Text] [PDF]
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 K. A. Leiner, D. Newman, C.-M. Li, E. Walsh, J. Khosla, and P. L. Sannes Heparin and Fibroblast Growth Factors Affect Surfactant Protein Gene Expression in Type II Cells Am. J. Respir. Cell Mol. Biol., November 1, 2006; 35(5): 611 - 618. [Abstract] [Full Text] [PDF]
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T. Warger, N. Hilf, G. Rechtsteiner, P. Haselmayer, D. M. Carrick, H. Jonuleit, P. von Landenberg, H.-G. Rammensee, C. V. Nicchitta, M. P. Radsak, et al. Interaction of TLR2 and TLR4 Ligands with the N-terminal Domain of Gp96 Amplifies Innate and Adaptive Immune Responses J. Biol. Chem., August 11, 2006; 281(32): 22545 - 22553. [Abstract] [Full Text] [PDF]
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 N. Sommerfeldt, P. Beckhove, Y. Ge, F. Schutz, C. Choi, M. Bucur, C. Domschke, C. Sohn, A. Schneeweis, J. Rom, et al. Heparanase: a new metastasis-associated antigen recognized in breast cancer patients by spontaneously induced memory T lymphocytes. Cancer Res., August 1, 2006; 66(15): 7716 - 7723. [Abstract] [Full Text] [PDF]
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K. A. Scheibner, M. A. Lutz, S. Boodoo, M. J. Fenton, J. D. Powell, and M. R. Horton Hyaluronan Fragments Act as an Endogenous Danger Signal by Engaging TLR2 J. Immunol., July 15, 2006; 177(2): 1272 - 1281. [Abstract] [Full Text] [PDF]
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 Z. Wang, L. Xiang, J. Shao, and Z. Yuan The 3' CCACCA Sequence of tRNAAla(UGC) Is the Motif That Is Important in Inducing Th1-Like Immune Response, and This Motif Can Be Recognized by Toll-Like Receptor 3 Clin. Vaccine Immunol., July 1, 2006; 13(7): 733 - 739. [Abstract] [Full Text] [PDF]
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 S. F. Hussain, D. Yang, D. Suki, K. Aldape, E. Grimm, and A. B. Heimberger The role of human glioma-infiltrating microglia/macrophages in mediating antitumor immune responses Neuro Oncology, July 1, 2006; 8(3): 261 - 279. [Abstract] [Full Text] [PDF]
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K. Izuishi, A. Tsung, G. Jeyabalan, N. D. Critchlow, J. Li, K. J. Tracey, R. A. Demarco, M. T. Lotze, M. P. Fink, D. A. Geller, et al. Cutting Edge: High-Mobility Group Box 1 Preconditioning Protects against Liver Ischemia-Reperfusion Injury. J. Immunol., June 15, 2006; 176(12): 7154 - 7158. [Abstract] [Full Text] [PDF]
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Y. Zhai, L. Meng, F. Gao, Y. Wang, R. W. Busuttil, and J. W. Kupiec-Weglinski CD4+ T Regulatory Cell Induction and Function in Transplant Recipients after CD154 Blockade Is TLR4 Independent J. Immunol., May 15, 2006; 176(10): 5988 - 5994. [Abstract] [Full Text] [PDF]
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 D. Bischof, S. F. Elsawa, G. Mantchev, J. Yoon, G. E. Michels, A. Nilson, S. L. Sutor, J. L. Platt, S. M. Ansell, G. von Bulow, et al. Selective activation of TACI by syndecan-2 Blood, April 15, 2006; 107(8): 3235 - 3242. [Abstract] [Full Text] [PDF]
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 A. Tedgui and Z. Mallat Cytokines in Atherosclerosis: Pathogenic and Regulatory Pathways Physiol Rev, April 1, 2006; 86(2): 515 - 581. [Abstract] [Full Text] [PDF]
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 G. J. Brunn, S. Saadi, and J. L. Platt Differential Regulation of Endothelial Cell Activation by Complement and Interleukin 1{alpha} Circ. Res., March 31, 2006; 98(6): 793 - 800. [Abstract] [Full Text] [PDF]
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E. F. Cohn, C. Nathan, D. Radzioch, H. Yu, Z. Xiang, and A. Ding Abrupt Expression of TLR4 in TLR4-Deficient Macrophages Imposes a Selective Disadvantage: Genetic Evidence for TLR4-Dependent Responses to Endogenous, Nonmicrobial Stimuli J. Immunol., January 15, 2006; 176(2): 1185 - 1194. [Abstract] [Full Text] [PDF]
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 K. R. Taylor and R. L. Gallo Glycosaminoglycans and their proteoglycans: host-associated molecular patterns for initiation and modulation of inflammation FASEB J, January 1, 2006; 20(1): 9 - 22. [Abstract] [Full Text] [PDF]
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 C. W. Frevert and P. L. Sannes Matrix proteoglycans as effector molecules for epithelial cell function Eur. Respir. Rev., December 1, 2005; 14(97): 137 - 144. [Abstract] [Full Text] [PDF]
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A. Tsung, R. A. Hoffman, K. Izuishi, N. D. Critchlow, A. Nakao, M. H. Chan, M. T. Lotze, D. A. Geller, and T. R. Billiar Hepatic Ischemia/Reperfusion Injury Involves Functional TLR4 Signaling in Nonparenchymal Cells J. Immunol., December 1, 2005; 175(11): 7661 - 7668. [Abstract] [Full Text] [PDF]
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T R D J Radstake, A W T van Lieshout, P L C M van Riel, W B van den Berg, and G J Adema Dendritic cells, Fc{gamma} receptors, and Toll-like receptors: potential allies in the battle against rheumatoid arthritis Ann Rheum Dis, November 1, 2005; 64(11): 1532 - 1538. [Abstract] [Full Text] [PDF]
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 K. Vermaelen and R. Pauwels Pulmonary Dendritic Cells Am. J. Respir. Crit. Care Med., September 1, 2005; 172(5): 530 - 551. [Abstract] [Full Text] [PDF]
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F. Geisler, H. Algul, M. Riemann, and R. M. Schmid Questioning Current Concepts in Acute Pancreatitis: Endotoxin Contamination of Porcine Pancreatic Elastase Is Responsible for Experimental Pancreatitis-Associated Distant Organ Failure J. Immunol., May 15, 2005; 174(10): 6431 - 6439. [Abstract] [Full Text] [PDF]
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 L. Bousarghin, P. Hubert, E. Franzen, N. Jacobs, J. Boniver, and P. Delvenne Human papillomavirus 16 virus-like particles use heparan sulfates to bind dendritic cells and colocalize with langerin in Langerhans cells J. Gen. Virol., May 1, 2005; 86(5): 1297 - 1305. [Abstract] [Full Text] [PDF]
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A. Tsung, R. Sahai, H. Tanaka, A. Nakao, M. P. Fink, M. T. Lotze, H. Yang, J. Li, K. J. Tracey, D. A. Geller, et al. The nuclear factor HMGB1 mediates hepatic injury after murine liver ischemia-reperfusion J. Exp. Med., April 4, 2005; 201(7): 1135 - 1143. [Abstract] [Full Text] [PDF]
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D. De Nardo, P. Masendycz, S. Ho, M. Cross, A. J. Fleetwood, E. C. Reynolds, J. A. Hamilton, and G. M. Scholz A Central Role for the Hsp90{middle dot}Cdc37 Molecular Chaperone Module in Interleukin-1 Receptor-associated-kinase-dependent Signaling by Toll-like Receptors J. Biol. Chem., March 18, 2005; 280(11): 9813 - 9822. [Abstract] [Full Text] [PDF]
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P. Tobias and L. K. Curtiss Thematic review series: The Immune System and Atherogenesis. Paying the price for pathogen protection: toll receptors in atherogenesis J. Lipid Res., March 1, 2005; 46(3): 404 - 411. [Abstract] [Full Text] [PDF]
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Y. Zhai, X.-d. Shen, R. O'Connell, F. Gao, C. Lassman, R. W. Busuttil, G. Cheng, and J. W. Kupiec-Weglinski Cutting Edge: TLR4 Activation Mediates Liver Ischemia/Reperfusion Inflammatory Response via IFN Regulatory Factor 3-Dependent MyD88-Independent Pathway J. Immunol., December 15, 2004; 173(12): 7115 - 7119. [Abstract] [Full Text] [PDF]
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M.-F. Tsan and B. Gao Endogenous ligands of Toll-like receptors J. Leukoc. Biol., September 1, 2004; 76(3): 514 - 519. [Abstract] [Full Text] [PDF]
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J. A. Whitsett, C. J. Bachurski, K. C. Barnes, P. A. Bunn Jr., L. M. Case, D. N. Cook, D. Crooks, M. W. Duncan, L. Dwyer-Nield, R. C. Elston, et al. Functional Genomics of Lung Disease Am. J. Respir. Cell Mol. Biol., August 1, 2004; 31(2/S1): S1 - S81. [Full Text] [PDF]
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 C. Farina, D. Theil, B. Semlinger, R. Hohlfeld, and E. Meinl Distinct responses of monocytes to Toll-like receptor ligands and inflammatory cytokines Int. Immunol., June 1, 2004; 16(6): 799 - 809. [Abstract] [Full Text] [PDF]
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K. R. Taylor, J. M. Trowbridge, J. A. Rudisill, C. C. Termeer, J. C. Simon, and R. L. Gallo Hyaluronan Fragments Stimulate Endothelial Recognition of Injury through TLR4 J. Biol. Chem., April 23, 2004; 279(17): 17079 - 17084. [Abstract] [Full Text] [PDF]
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 H.-J. Anders, B. Banas, and D. Schlondorff Signaling Danger: Toll-Like Receptors and their Potential Roles in Kidney Disease J. Am. Soc. Nephrol., April 1, 2004; 15(4): 854 - 867. [Abstract] [Full Text] [PDF]
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K. Kariko, H. Ni, J. Capodici, M. Lamphier, and D. Weissman mRNA Is an Endogenous Ligand for Toll-like Receptor 3 J. Biol. Chem., March 26, 2004; 279(13): 12542 - 12550. [Abstract] [Full Text] [PDF]
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J.-L. Imler and L. Zheng Biology of Toll receptors: lessons from insects and mammals J. Leukoc. Biol., January 1, 2004; 75(1): 18 - 26. [Abstract] [Full Text] [PDF]
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G. B. Johnson, G. J. Brunn, and J. L. Platt Cutting Edge: An Endogenous Pathway to Systemic Inflammatory Response Syndrome (SIRS)-Like Reactions through Toll-Like Receptor 4 J. Immunol., January 1, 2004; 172(1): 20 - 24. [Abstract] [Full Text] [PDF]
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I. Caramalho, T. Lopes-Carvalho, D. Ostler, S. Zelenay, M. Haury, and J. Demengeot Regulatory T Cells Selectively Express Toll-like Receptors and Are Activated by Lipopolysaccharide J. Exp. Med., February 17, 2003; 197(4): 403 - 411. [Abstract] [Full Text] [PDF]
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M. E. Mummert, D. Mummert, D. Edelbaum, F. Hui, H. Matsue, and A. Takashima Synthesis and Surface Expression of Hyaluronan by Dendritic Cells and Its Potential Role in Antigen Presentation J. Immunol., October 15, 2002; 169(8): 4322 - 4331. [Abstract] [Full Text] [PDF]
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