Lipopolysaccharide induces expression of fibronectin
5
1-integrin receptors in human monocytic cells in a protein kinase C-dependent fashion
Jesse Roman,
Jeffrey D. Ritzenthaler,
Bonnie Boles,
Manuel Lois, and
Susanne Roser-Page
Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Emory University School of Medicine, and The Atlanta Veterans Affairs Medical Center, Atlanta, Georgia 30322
Submitted 22 July 2003
; accepted in final form 24 March 2004
 |
ABSTRACT
|
---|
LPS is an outer-membrane glycolipid component of gram-negative bacteria known for its fervent ability to activate monocytic cells and for its potent proinflammatory capabilities. In addition, LPS triggers the release of cytokines and chemokines as well as cell-cell adhesion molecules. We postulate that LPS may also affect the expression of matrix-binding integrin receptors, thereby modulating cell-adhesive functions in monocytic cells. To test this hypothesis, we investigated the effects of LPS on the expression of the integrin
5
1, a fibronectin receptor, in a human monocytic cell line (U937) as well as in isolated human peripheral blood mononuclear cells (PBMCs). We found that LPS increased the expression of
5
1 receptors and enhanced the adherence of U937 cells and PBMCs to fibronectin-coated surfaces; this was blocked by anti-
5
1 antibodies. LPS increased
5-subunit mRNA accumulation in a dose- and time-dependent manner. The induction by LPS occurred, at least in part, at the level of gene transcription as indicated by experiments using
5 intact and deletion promoter constructs. LPS-induced
5 gene transcription was associated with rapid induction of conventional PKC-
protein and activity, was blocked by PKC inhibitors, and was mimicked by lipid A. Finally, we found that an anti-CD14 antibody was able to inhibit the LPS response. Overall, the data suggest that LPS stimulates
5 gene transcription via CD14 and PKC-dependent signals to enhance the expression of functional
5
1 receptors in monocytic cells. This process may help stimulate monocytic cell activation and facilitate their migration into fibronectin-containing tissues during infection.
integrins; sepsis; endotoxin; signal transduction
THE INFECTION OF TISSUES with gram-negative bacteria triggers a cascade of events that help establish inflammation. If left uncontrolled, infection can lead to grave consequences, including hypotension, disseminated intravascular coagulation, acute respiratory distress syndrome, multiorgan failure, and death (13). The pathophysiological mechanisms that aid in establishing inflammation during gram-negative bacterial infection are not entirely understood. It is known, however, that the interaction between bacteria or bacterial products with host cells (e.g., monocytic cells/macrophages, polymorphonuclear cells, and endothelial cells) is one of the first steps involved in the elicitation of host cell-derived factors (e.g., chemokines, cytokines, eucosanoids) responsible for the initiation and amplification of the inflammatory response (13, 28, 32).
One bacterial product known for its ability to activate monocytic cells during gram-negative bacterial infection is lipopolysaccharide (LPS). LPSs are immunogenic glycolipids that make up the outer portion of the outer membrane of gram-negative bacteria (32, 47). There are three major domains that comprise the LPS molecule: the lipid A domain (also known as endotoxin), which functions to anchor LPS in the outer membrane and is responsible for the severe systemic inflammatory response associated with severe gram-negative infections; the core domain, made of phosphorylated nonrepeating oligosaccharides that help form the outer membrane and serve as a barrier to antibiotics; and the O-antigen polymer domain, composed of varying lengths of immunogenic repeating oligosaccharides (32). The stimulatory effects of LPS on monocytic cells appear to be elicited by the binding of lipid A to specific surface receptors expressed by the immune cells termed CD14 (11, 31, 50, 51). Binding to CD14 is facilitated by a specific protein termed lipid A-binding protein (LBP) that is produced by hepatocytes in response to cytokines (42, 47, 48). CD14, in conjunction with LBP and MD-2 accessory protein, presents LPS to a coreceptor (18). This CD14 coreceptor is a member of a growing family of Toll-like receptor proteins (TLRs) responsible for LPS intracellular signaling (18). Once activated, these receptors trigger a number of host responses by mechanisms that remain poorly defined.
An important aspect of the inflammatory response against gram-negative bacteria is the recruitment of immune cells into the affected tissues. This process is dependent on cell-cell adhesion events that mediate the transfer of immune cells from the intravascular space into the interstitium, followed by cell-matrix events responsible for the migration of immune cells into tissues and toward the site of infection (35, 44). In the absence of cell-cell adhesion events, immune cell delivery to infected tissues is inhibited, resulting in unopposed infection. This is observed in children with leukocyte adhesion deficiency, a disease characterized by the absence or malfunction of
2-integrin receptors that mediate the interaction between immune cells and the endothelium (3). The role of cell-matrix interactions is considered equally important, but the mechanisms that control these processes are inadequately understood (35).
This report explores the mechanisms by which LPS stimulates the adhesion of monocytic cells to fibronectin, a matrix glycoprotein highly expressed in injured tissues (33, 35, 36). Many of the cellular effects of fibronectin are mediated via the integrin
5
1, a heterodimeric transmembrane glycoprotein capable of signal transduction (33, 37, 40). The interaction of immune cells with fibronectin promotes not only cell adhesion and migration/chemotaxis but also cell activation and the production of proinflammatory cytokines (1, 37, 40). These events are elicited by fibronectin-induced, integrin-mediated signals that induce potent transcription factors (e.g., activator protein-1 and NF-
B) capable of stimulating the transcription of gene products that affect host inflammatory responses (7, 33, 38). Herein, we demonstrate that LPS can modulate monocytic cell adhesion to fibronectin by affecting the transcription of the
5-integrin gene, thereby stimulating the expression of functional fibronectin
5
1 receptors.
 |
METHODS
|
---|
Reagents.
MEK1 inhibitor PD-98059 was purchased from New England Biolabs (Beverly, MA). The anti-
2 antibody (P1E6; directed to the
2-subunit of the
2
1-integrin), the anti-
5 antibody (PID6; directed to the
5-subunit of the
5
1-integrin), and the anti-
1 antibody (P4C10; directed to the
1-integrin subunit) were purchased from Life Technologies (Gaithersburg, MD). The anti-
2 antibody (MAB1962) was purchased from Chemicon (Temecula, CA). The anti-CD14 antibody (AB383) was purchased from R&D (Minneapolis, MN). All other reagents were purchased from Sigma Chemical (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA) unless otherwise specified.
Cell culture and treatment.
Human monocytic/macrophage cells from histiocytic lymphoma (U937, ATCC CRL no. 1593.2) were maintained in RPMI 1640 (MediaTech, Herndon, VA) supplemented with 10% heat-inactivated FBS and 1% antiobiotic-antimycotic solution (100 U/ml penicillin G sodium, 100 U/ml streptomycin sulfate, and 0.25 µg/ml amphotericin B) and incubated in a humidified 5% CO2 incubator at 37°C. Human peripheral blood mononuclear cells (PBMCs) from healthy, nonsmoking volunteers were isolated by elutriation on a polysucrose diatrizoate column (Histopaque-1077) under sterile conditions as previously described (14). Cells were judged to be >95% viable by trypan blue exclusion.
Cell adhesion assay.
The adhesion of U937 cells or PBMCs (1 x 105 cells) was tested on 96-well fibronectin-coated (50 µg/ml) polystyrene plates as previously described (36). Cells were exposed to LPS (5 µg/ml) for 3 h at 37°C, washed, and added to fibronectin-coated plates. Nonadherent cells were washed from the dishes after 1 h, and the remaining adherent cells were quantified by using a colorimetric-type assay that detects the intracellular enzyme hexosaminidase as described by Landegren (19). To determine the role of the
5
1-integrin in cell adhesion to fibronectin, the cells were pretreated with anti-
5 antibody (PID6, 100 µg/ml), anti-
1 antibody (P4C10, 100 µg/ml), anti-
2 antibody (MAB1962, 100 µg/ml), or a control IgG for 30 min before adhesion to fibronectin-coated dishes.
RNA isolation and real-time PCR analysis.
Total cellular RNA was extracted from U-937 cells and PBMCs using a published method (8) and modified as we previously described (29). The reverse transcription reactions of the extracted RNA were performed by combining the following reagents in a PCR reaction tube: 0.625 mM dNTP, 16 nmol of random hexamer oligonucleotides (Boehringer Mannheim no. 1277049), 5 µl of first strand buffer (50 mM Tris·HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2; Life Technologies), 20 mM DTT, 200 units of reverse transcriptase enzyme, 0.5 µl of RNasin (ribonuclease inhibitor; Promega, Madison, WI), and 1 µg of extracted RNA in a total volume of 25 µl. Samples were heated to 70°C for 5 min, chilled on ice for 5 min, centrifuged briefly, incubated at 42°C for 1 h followed by a 10-min incubation at 90°C, and chilled on ice for 5 min.
Real-time PCR reactions were set up by adding the following reagents to Smart Cycler reaction tubes (Sunnyvale, CA): 5 mM MgCl2, 0.2 µM forward primer, 0.2 µM reverse primer, 10x Master Mix (LightCycler FastStart Master SYBR Green I; Roche, Indianapolis, IN), and template cDNA (500 ng total). Forward and reverse primers for human
5 mRNA were 5'-AgCCgCAgCTCTgCTTC and 5'-AgCTgTggCCACCTgAC, respectively. Forward and reverse primers for human
-actin mRNA were 5'-TggAgAAAATCTggCACC and 5'-gTATggggAgCATCTACC, respectively. Samples were briefly centrifuged and processed using the following cycle program with the Cepheid Smart Cycler: hold at 95°C for 600 s followed by 40 cycles at temperatures of 95°C for 15 s, 68°C for 30 s (with Optics on), and 72°C for 30 s. Results of the log-linear phase of the growth curve were analyzed by use of the mathematical equation of the second derivative, and relative quantification was performed using the 2-
CT method (22).
Removal of LPS.
LPS was removed from samples by an endotoxin affinity resin (Associates of Cape Cod, Woods Hole, MA) following the manufacturer's instructions. All samples were tested after LPS removal (30) and determined to contain <0.06 ng/ml of LPS, which is within the accepted background levels of other endotoxin assays.
Detection of
5-integrin and PKC-
by Western blot.
U-937 cells (1 x 106 cells/ml) were treated with 5 µg/ml of LPS for 48 h (
5-integrin) or for 4 h (PKC-
), washed with ice-cold PBS, and lysed in 1 ml of homogenization buffer (50 mM NaCl, 50 mM NaF, 50 mM NaP2O7-10 H2O, 5 mM EDTA, 5 mM EGTA, 2 mM Na3V04, 0.5 mM PMSF, 0.01% Triton X-100, 10 µg/ml leupeptin, and 10 mM HEPES, pH 7.4) by repeated passages through a 26-gauge needle. The resulting homogenate was centrifuged at 14,000 rpm for 5 min at 4°C. Protein concentration was determined by the Bradford method (5). The protein (100 µg) was mixed with an equal volume of 2x sample buffer (125 mM Tris·HCl, pH 6.8, 4% SDS, 20% glycerol, 510% 2-mercaptoethanol, and 0.004% bromphenol blue), boiled for 5 min, loaded onto a 7% (
5-integrin) or a 10% (PKC-
) SDS-polyacrylamide gel with a 5% stacking gel, and electrophoresed for 2 h at 60 mA. The separated proteins were transferred onto nitrocellulose using a Bio-Rad Trans Blot semidry transfer apparatus for 1 h at 25 mA, blocked with Blotto [1x TBS (10 mM Tris·HCl, pH 8.0, 150 mM NaCl), 5% nonfat dry milk, 0.05% Tween 20] for 1 h at room temperature, and washed twice for 5 min with wash buffer (1x TBS, 0.05% Tween 20). Blots were incubated with a polyclonal antibody raised against human
5-integrin (antibody sc-6595; 1:500 dilution) or an antibody against the COOH-terminal region of PKC-
(Sigma antibody P4334; 1:3,000 dilution) for 24 h at 4°C, washed three times for 5 min with wash buffer, and incubated with a secondary rabbit antibody raised against goat IgG conjugated to horseradish peroxidase (1:20,000 dilution) for 1 h at room temperature. Identically loaded blots used for loading controls were incubated with either
-actin (abcam 1801; 1:1,000 dilution) or GAPDH (abcam 9485; 1:2,000 dilution) primary antibodies. Blots were washed four times for 5 min in wash buffer, transferred to freshly made enhanced chemiluminescence solution (Amersham, Arlington, IL) for 5 min, and exposed to X-ray film. Protein bands were quantified by densitometric scanning using a GS-800 calibrated laser densitometer (Bio-Rad, Hercules, CA).
PKC activity.
PKC activity was measured by using the Kinase-Glo Luminescent kinase assay method (Promega) following the manufacturer's instructions. Briefly, U937 cells (1 x 107) were grown in suspension at 37°C in a 5% CO2 incubator in the presence or absence of 5 µg/ml of LPS for 4 h. Cells were harvested, washed with ice-cold PBS, resuspended in 200 µl of kinase reaction buffer (40 mM Tris, pH 7.5, 20 mM MgCl2, 0.1 mg/ml BSA), and sonicated. Samples were diluted in 50 µl of PKC reaction buffer (20 mM Tris, pH 7.5, 10 mM MgCl2, 0.1 mg/ml BSA, 250 µM EGTA, 400 µM CaCl2, 0.32 mg/ml phosphatidylserine, 0.032 mg/ml diacylglycerol) with 10 µM ATP and 100 µM neurogranin(2843) for 90 min at room temperature. Kinase-Glo reagent (50 µl) was added, samples were incubated at room temperature for 10 min, and light intensity was measured using a ThermoLabsystems Luminoskan Ascent microtiter plate luminometer. Results were recorded as inverse relative light units. Protein concentrations were determined by the Bradford method using Bio-Rad protein assay reagent (5).
Electroporation and luciferase assays.
Electroporation of U937 cells was used to introduce the
5 promoter constructs: p
5(938 bp)LUC, p
5(178 bp)LUC, p
5(92 bp)LUC, p
5(41 bp)LUC, p
5(27 bp)LUC, and p
5(1 bp)LUC as previously described (4). Briefly, cells were washed with PBS and added to serum-free media supplemented with 10 mM dextrose and 0.1 mM DTT to a final concentration of 6 x 107 cells/ml. U937 cells (4.8 x 107 cells) were added to electroporation cuvettes (0.4-cm electrode gap) along with 40 µg of promoter construct plasmid DNA and 20 µg of the
-galactosidase reporter plasmid DNA and were subjected to 400 V and 1075 µF (Gene Pulser II Electroporation System, Bio-Rad). Electroporated cells were pooled, aliquoted into 24-well plates, and incubated with or without LPS (010 µg/ml) for 3 h at 37°C and 5% CO2. In antibody studies, cells were preincubated for 1 h at 37°C and 5% CO2 with anti-
2, anti-
5, anti-
1, anti-
2, anti-CD14 antibody, or control antibodies. Cells were harvested, washed, and resuspended in 100 µl of cell lysis buffer, and a 20-µl aliquot was tested for luciferase activity by adding 50 µl of luciferase assay reagent (Promega). Light intensity was measured using a ThermoLabsystems Luminoskan Ascent microtiter plate luminometer. Results were recorded as relative luciferase units and standardized for transfection efficiency using
-galactosidase activity.
EMSA.
U-937 cells (1 x 108) were grown in suspension at 37°C in a 5% CO2 incubator in the presence of 5 µg/ml of LPS for 20 h. Cells were washed with ice-cold PBS, and nuclear binding proteins were extracted by a published method (9). Protein concentrations were determined by the Bradford method using Bio-Rad protein assay reagent (5). Double-stranded NF-
B or AP-1 consensus oligonucleotides were radiolabeled with [
-32P]ATP using T4 polynucleotide kinase enzyme. Nuclear protein (5 µg) was incubated with radiolabeled NF-
B or AP-1 (50100,000 cpm/ng) for 30 min at room temperature as described previously (33). For competition reactions, 50-fold molar excess of double-stranded NF-
B (5'-AgTTgAggggACTTTCCCAggC) consensus oligonucleotide or AP-1 consensus oligonucleotide (5'-CgCTTgATgACTCAgCCggAA) or double-stranded mutated NF-
B oligonucleotide (5'-AgTTgAggCgACTTTCCCAggC) or AP-1 mutated oligonucleotide (5'-CgCTTgATgACTTggCCggAA) was added to the reaction. DNA-protein complexes were separated on 6% native polyacrylamide gel (20:1 acrylamide/bis ratio) in low ionic strength buffer (22.25 mM Tris borate, 22.25 mM boric acid, 500 mM EDTA) for 23 h at 4°C at 10 V/cm. Gels were fixed in a 10% acid acid/10% methanol solution for 10 min, dried under vacuum, and exposed to X-ray film. Radiolabeled DNA-protein complexes were extracted from gels and quantified by a scintillation counter.
Statistical evaluation.
Means ± SD were calculated for all experimental values. Significance was assessed by ANOVA followed by Student's t-test. All experiments were repeated 49 times.
 |
RESULTS
|
---|
LPS enhances U937 cell and PBMC adhesion to fibronectin via
5
1.
To determine whether LPS could enhance the adhesion of monocytic cells to fibronectin, U937 cells were exposed to 5 µg/ml of LPS for 3 h and submitted to an adhesion assay using 96-well tissue culture plates coated with fibronectin (50 µg/ml). As shown in Fig. 1A, LPS significantly increased (P < 0.001) the adhesion of U937 cells to fibronectin. This increase in cell adhesion to fibronectin after LPS treatment was associated with an increase in
5
1-integrin protein in U937 cells as demonstrated by Western blot in Fig. 1B. Freshly isolated PBMCs also showed a similar increase in the amount of
5
1-integrin protein in response to LPS treatment compared with nonstimulated control cells (Fig. 1B). To further define the role of the
5
1-integrin in constitutive and LPS-induced adhesion to fibronectin-coated plates, U937 cells and PBMCs were pretreated with blocking anti-
5-, anti-
2-, or anti-
1-integrin antibodies before submission to an adhesion assay. As expected, the increased cell adhesion to fibronectin after exposure to LPS was blocked by the anti-
5- and anti-
1-integrin antibodies; however, an antibody against the
2-integrin subunit failed to block the adhesion of cells to fibronectin-coated plates as shown in Fig. 1.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 1. Integrin 5 1 receptors are involved in LPS-stimulated adhesion to fibronectin. A: U937 cells (1 x 105 cells) were exposed to LPS (5 µg/ml) for 3 h at 37°C, washed, and added to fibronectin-coated (50 µg/ml) polystyrene plates for 1 h at 37°C. Nonadherent cells were washed from the plates, and the remaining adherent cells were quantified by using a colorimetric-type assay that detects the intracellular enzyme hexosaminidase (19). Data are presented as means ± SD (n = 9). Note that LPS significantly increased the adhesion of U937 cells (P < 0.001) to fibronectin-coated plates. B: U937 cells or peripheral blood mononuclear cells (PBMCs; 1 x 106 cells/ml) were treated with 5 µg/ml of LPS for 48 h, washed with ice-cold PBS, and lysed in 1 ml of homogenization buffer. Proteins (100 µg) were separated on a 7% SDS-polyacrylamide gel, transferred onto nitrocellulose, and incubated with an anti-human 5-integrin antibody (1:500 dilution) for 24 h at 4°C. -Actin antibody (1:1,000 dilution) was used to control for loading. The blots were incubated with a secondary rabbit anti-goat IgG conjugated to horseradish peroxidase, transferred to freshly made enhanced chemiluminescence solution, and exposed to X-ray film. Protein bands were quantified by densitometric scanning using a GS-800 calibrated laser densitometer (Bio-Rad; n = 4). C: U937 cells (1 x 105 cells) were preincubated with anti- 5 antibody (PID6, 100 µg/ml), anti- 1 antibody (P4C10, 100 µg/ml), anti- 2 antibody (MAB1962, 100 µg/ml), or a control IgG (not shown) for 30 min before exposure to LPS (5 µg/ml) for 3 h at 37°C. Cells were added to fibronectin-coated (50 µg/ml) polystyrene plates for 1 h at 37°C. Nonadherent cells were washed from the plates, and the remaining adherent cells were quantified by using the colorimetric-type assay. Data are presented as means ± SD (n = 9). Note that LPS significantly increased the adhesion of U937 cells (P < 0.001) to fibronectin-coated plates and that pretreatment with anti- 5- and anti- 1-integrin antibodies blocked this adhesion (anti- 5, P < 0.001; anti- 1, P < 0.004). Inset: standard curve for U937 adhesion to fibronectin-coated polystyrene plates; r = 0.996. D: PBMCs (1 x 105 cells) were preincubated with anti- 5 antibody (PID6, 100 µg/ml), anti- 1 antibody (P4C10, 100 µg/ml), anti- 2 antibody (MAB1962, 100 µg/ml), or a control IgG (not shown) for 30 min before exposure to LPS (5 µg/ml) for 3 h at 37°C. Cells were added to fibronectin-coated (50 µg/ml) polystyrene plates for 1 h at 37°C. Nonadherent cells were washed from the plates, and the remaining adherent cells were quantified by using the colorimetric-type assay. Data are presented as means ± SD (n = 4). Note that LPS significantly increased the adhesion of PBMCs (P < 0.001) to fibronectin-coated plates and that pretreatment with anti- 5- and anti- 1-integrin antibodies blocked this adhesion (P < 0.001). Inset: standard curve for PBMC adhesion to fibronectin-coated polystyrene plates; r = 0.988.
|
|
LPS induces transcription of the
5-integrin mRNA.
Because increased adhesion of U937 cells and PBMCs to fibronectin after LPS treatment was associated with an increase in the amount of
5
1-integrins present in these cells, real-time PCR was used to determine whether this coincided with an increase in endogenous mRNA accumulation for the
5-integrin. We found that the increase in the
5 mRNA was both time and dose dependent. In Fig. 2A,
5-integrin mRNA expression was measured after cells were exposed to increasing doses of LPS, ranging from 500 ng/ml to 10 µg/ml. LPS had the greatest effect on
5-integrin mRNA levels at doses between 1 and 5 µg/ml with cycle threshold (CT) values of 25.19 (P < 0.006) and 26.66 (P < 0.001), respectively, compared with nontreated control cells (CT = 28.31). In Fig. 2B, the effect of LPS (5 µg/ml) was optimal at 8 h (CT = 14.18, P < 0.005) compared with nontreated control cells (CT = 18.10). Similar results were detected in PBMCs as demonstrated in Fig. 2C, with threshold values of 29.0 and 30.56 for LPS-treated cells and nontreated control cells, respectively (P < 0.01).
LPS induces transcription of the
5-integrin gene.
The data presented above demonstrate that LPS induces the expression of the
5
1-integrin in U937 cells and PBMCs. To determine whether the stimulatory effect of LPS occurs at the level of gene transcription, experiments were performed in U937 cells transiently transfected with the human
5 gene promoter connected to a luciferase reporter gene. Transfected cells were stimulated with 515 µg/ml of LPS for 320 h at 37°C and 5% CO2. The cells were harvested, and an aliquot of cell extract was used to measure luciferase activity. We observed that LPS indeed induced the transcription of the
5 promoter construct, which was maximal at a concentration of 5 µg/ml of LPS and after 20 h of incubation (Fig. 3A). To determine the functional significance of LPS in the induction of the
5 promoter, LPS was removed from the cell culture medium using an endotoxin affinity resin. After LPS removal, the medium was determined to contain <0.06 ng/ml of LPS. This resulted in a significant inhibition (P < 0.001) of the LPS-induced transcription of the
5 gene (Fig. 3B).
Induction of
5 gene expression by LPS is dependent on PKC activation.
Because LPS has been previously shown to induce the activation of PKC, we set out to investigate whether its stimulatory effect on
5 gene transcription was mediated by this pathway. First, in support for a role of PKC in LPS induction of
5, we demonstrated in Fig. 4A that LPS treatment of U-937 cells enhanced the expression of conventional PKC-
(cPKC-
) protein, which was maximal at 4 h. cPKC-
is one isoform of PKC shown to regulate selective LPS-induced macrophage functions involved in the host defense and inflammation response (32). Second, to confirm the function of the enhanced expression of PKC by LPS, we measured active PKC with the use of a luminescent kinase assay. U937 cells treated with LPS showed a significant increase (P < 0.001) in PKC activity compared with nontreated control cells as demonstrated in Fig. 4B. Third, we showed that pretreatment of U-937 cells with active calphostin C (CC*) or chelerythrine chloride, two potent inhibitors of PKC, abrogated the increase in
5 gene transcription in response to LPS treatment (Fig. 4, C and D, respectively). In contrast, inactivated PKC inhibitor (CC) did not affect
5 expression (Fig. 4C).

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 4. Role of conventional PKC- (cPKC- ) in LPS induction of the 5 gene. A: U937 cells (1 x 106 cells/ml) were treated with 5 µg/ml of LPS for 4 h, washed with ice-cold PBS, lysed, and processed for Western blot with a polyclonal antibody raised against human cPKC- (1:3,000 dilution). GAPDH antibody (1:2,000 dilution) was used to control for loading. Protein bands were quantified by densitometric scanning using a laser densitometer. B: PKC activity was measured in U937 cells (1 x 107) incubated at 37°C and 5% CO2 in the presence or absence of 5 µg/ml of LPS for 4 h. Cells were harvested, washed, resuspended kinase reaction buffer, and sonicated. Samples were diluted in 50 µl of PKC reaction buffer and incubated for 90 min at room temperature. Kinase-Glo reagent (50 µl) was added, samples were incubated at room temperature for 10 min, and light intensity was measured using a ThermoLabsystems Luminoskan Ascent microtiter plate luminometer. Results were recorded as inverse relative light units/µg protein (n = 4). Protein concentrations were determined by the Bradford method using Bio-Rad protein assay reagent (5). Note that U937 cells treated with LPS demonstrated a significant increase in PKC activity (P < 0.001) compared with nontreated control cells. C: U937 cells (6 x 107 cells/ml) were electroporated with the human 5 promoter p 5(938 bp)LUC, washed, pooled, and aliquoted into 24-well plates. Cells were treated with or without LPS (5 µg/ml) for 20 h at 37°C and 5% CO2 in the presence or absence of the PKC inhibitor calphostin C (CC, inactive; CC*, active) followed by testing for luciferase activity. Light intensity was measured using a plate luminometer, and the results were recorded as relative luciferase units and standardized for transfection efficiency using -galactosidase activity. Data are presented as means ± SD (n = 9). Note that CC* (1 x 107 M) completely abrogated the LPS-induced 5 gene transcription (P < 0.001). D: U937 cells (6 x 107 cells/ml) were electroporated with the human 5 promoter p 5(938 bp)LUC, washed, pooled, and aliquoted into 24-well plates. Cells were treated with or without LPS (5 µg/ml) for 20 h at 37°C and 5% CO2 in the presence or absence of chelerythrine chloride (CHE; 1 µM) followed by testing for luciferase activity. Light intensity was measured using a plate luminometer, and the results were recorded as relative luciferase units and standardized for transfection efficiency using -galactosidase activity. Data are presented as means ± SD (n = 6). Note that CHE abrogated the LPS-induced 5 gene transcription (P < 0.001).
|
|
Transcriptional regulation of the
5 gene in response to LPS.
To learn more about the possible cis-acting elements involved in the LPS stimulation of the
5 gene transcription, U937 cells were transfected with the 923-bp
5 promoter construct p
5(938 bp)LUC or various 5' deletion promoter constructs: p
5(178 bp)LUC, p
5(92 bp)LUC, p
5(41 bp)LUC, p
5(27 bp)LUC, and p
5(1 bp)LUC (Fig. 5). As before, LPS stimulated the expression of the p
5(923 bp)LUC promoter construct >3.7-fold compared with controls not stimulated with LPS. After the deletion of 805 bp of the distal end of the p
5(923 bp)LUC promoter to create the p
5(178 bp)LUC construct, the intensity of the LPS response diminished 1.12-fold compared with the p
5(923 bp)LUC construct. When an additional 86 bp of the 5' end of the p
5(178 bp)LUC promoter was deleted to make the p
5(0.92 bp)LUC construct, LPS stimulation of the
5 promoter, when analyzed against control, was reduced 2.19-fold compared with the p
5(923 bp)LUC construct. However, the most drastic reduction [>2.75-fold compared with the p
5(923 bp)LUC construct] in LPS-stimulated
5 promoter expression was seen with plasmids p
5(41 bp)LUC, p
5(26 bp)LUC, and p
5(1 bp)LUC. This indicates the presence of a key LPS response element(s) located in the
5 promoter between 92 and 41 bp downstream from the transcriptional start site. This promoter region also appears important for the basal expression of the
5 gene since there was very little promoter activity in cells transfected with the 27-bp and 1-bp deletion constructs.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 5. LPS responsive region is located between 92 and 41 bp downstream from the transcriptional start site of the 5 gene. U937 cells (6 x 107 cells/ml) were electroporated with the following human 5 promoter or deletion constructs: p 5(938 bp)LUC, p 5(178 bp)LUC, p 5(92 bp)LUC, p 5(41 bp)LUC, p 5(27 bp)LUC, and p 5(1 bp)LUC. Cells were treated with or without LPS (5 µg/ml) for 20 h at 37°C and 5% CO2, harvested, washed, and resuspended in 100 µl of cell lysis buffer, and a 20-µl aliquot was tested for luciferase activity. Results were recorded as relative luciferase units and standardized for transfection efficiency using -galactosidase activity. Data are presented as means ± SD (n = 9). Note that the LPS responsive region within the 5 promoter is located between 92 and 41 bp from the transcriptional start site. C, control.
|
|
Trans-acting factor NF-
B, but not AP-1, is involved in LPS induction of the
5 promoter.
Nuclear binding proteins were isolated from U937 cells stimulated with or without LPS for 20 h and subjected to an EMSA using radiolabeled consensus NF-
B and AP-1 oligonucleotides. Data shown in Fig. 6A show an increase in the bound NF-
B nuclear protein-DNA complex in cells treated with LPS compared with control untreated cells. To demonstrate DNA-protein binding specificity, nonradiolabeled competitor NF-
B or mutated NF-
B oligonucleotide (50-fold molar excess) was added to the binding reaction. The consensus NF-
B, but not the mutated NF-
B competitor, was able to compete for binding. There was also a slight increase in the bound AP-1 nuclear protein-DNA complex in LPS-treated cells (Fig. 6B). Again, to demonstrate DNA-protein binding specificity, nonradiolabeled competitor AP-1 or mutated AP-1 oligonucleotide (50-fold molar excess) was added to the binding reaction. To determine whether NF-
B binding proteins were involved in the function of transcriptional regulation of the
5 gene, consensus NF-
B competing oligonucleotides were cotransfected into U937 cells along with the p
5(923 bp)LUC promoter and stimulated with or without LPS. Data in Fig. 7A confirm that the NF-
B is an important cis-acting element for the LPS induction of the
5 gene since competing NF-
B is able to abrogate the LPS induction. Figure 7B, on the other hand, demonstrates that the AP-1 cis-acting element is not essential for the LPS induction of the
5 gene since it was unable to abrogate the LPS induction.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 6. Role of NF- B and AP-1 in LPS-induced 5 gene transcription. A: U937 cells (1 x 108) were grown in suspension at 37°C in a 5% CO2 incubator in the presence or absence of 5 µg/ml of LPS. Cells were washed, and nuclear binding proteins were extracted. Radiolabeled double-stranded NF- B consensus oligonucleotide (75100,000 cpm/ng) was incubated with extracted nuclear binding proteins for 30 min at room temperature. DNA-protein complexes were separated on 6% native polyacrylamide gel, gels were fixed in a 10% acid acid/10% methanol, dried, and exposed to X-ray film. Note that LPS induced more binding to NF- B compared with control nontreated cells and that the nonradiolabeled competitor NF- B, but not mutated NF- B oligonucleotide (50-fold molar excess), was able to compete for binding. B: U937 cells (1 x 108) were grown in suspension at 37°C in a 5% CO2 incubator in the presence or absence of 5 µg/ml of LPS. Cells were washed, and nuclear binding proteins were extracted. Radiolabeled double-stranded AP-1 consensus oligonucleotide (5070,000 cpm/ng) was incubated with extracted nuclear binding proteins for 30 min at room temperature. DNA-protein complexes were separated on 6% native polyacrylamide gel, gels were fixed in a 10% acid acid/10% methanol, dried, and exposed to X-ray film. Note that LPS induced slightly more binding to AP-1 compared with control nontreated cells and that the nonradiolabeled competitor AP-1, but not mutated AP-1 (mAP-1) oligonucleotide (50-fold molar excess), was able to compete for binding.
|
|

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 7. NF- B, but not AP-1, cis-acting element is involved in LPS-induced 5 gene transcription. A: U-937 cells (6 x 107 cells/ml) were electroporated with the full-length human 5 promoter p 5(938 bp)LUC, washed, pooled, and aliquoted into 24-well plates. Cells were treated with LPS (5 µg/ml) in the presence or absence of consensus NF- B oligonucleotide (20 µg) for 20 h at 37°C and 5% CO2. Cells were harvested, washed, resuspended in 100 µl of cell lysis buffer, and tested for luciferase activity. Results were recorded as relative luciferase units and standardized for transfection efficiency using -galactosidase activity. Data are presented as means ± SD (n = 9). Note that cotransfection with consensus NF- B oligonucleotide inhibited the LPS-induced response (P < 0.001). B: U937 cells (6 x 107 cells/ml) were electroporated with the full-length human 5 promoter p 5(938 bp)LUC, washed, pooled, and aliquoted into 24-well plates. Cells were treated with LPS (5 µg/ml) in the presence or absence of consensus AP-1 oligonucleotide (20 µg) for 20 h at 37°C and 5% CO2. Cells were harvested, washed, resuspended in 100 µl of cell lysis buffer, and tested for luciferase activity. Results were recorded as relative luciferase units and standardized for transfection efficiency using -galactosidase activity. Data are presented as means ± SD (n = 9). Note that cotransfection with consensus AP-1 oligonucleotide had no effect on the LPS-induced response.
|
|
Lipid A mimics the induction of the
5 gene transcription.
Because the lipid A portion of LPS is considered the key component responsible for the pathophysiology of severe gram-negative infections, we set out to determine whether lipid A could mimic the effect of intact LPS on the induction of the
5 gene. Again, U937 cells were transfected with the p
5(923 bp)LUC promoter construct and treated with or without the lipid A component, and
5 gene transcription was quantified by luminescence. As shown in Fig. 8, lipid A was identical to LPS in its ability to significantly induce
5 gene transcription (P < 0.05).
LPS induction involves the CD14 receptor.
Data from previous reports identify the CD14 receptor as being a key component in the LPS induction pathway, and to test its importance in our system, U937 cells transfected with the p
5(923 bp)LUC construct were pretreated with the anti-CD14 antibody and stimulated with or without LPS. Additional antibodies, such as Ab5 or control IgG, anti-integrin
5, anti-CD11b, and anti-integrin
2 were also used. As the data show in Fig. 9, pretreatment of cells with the various antibodies did not significantly alter the baseline induction of the
5 promoter. In contrast, pretreatment of cells with the anti-CD14 antibody completely blocked the LPS induction of the
5 gene transcription (P < 0.001). Treatment with Ab5 or control IgG and the anti-
2 antibody did not inhibit the LPS induction of the
5 promoter (P = 0.9 and P = 0.1, respectively), whereas an anti-CD11b slightly reduced the LPS effect (P < 0.01).

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 9. Anti-CD14 antibody inhibits the LPS induction of the 5 gene transcription. U937 cells (6 x 107 cells/ml) were electroporated with the full-length human 5 promoter p 5(938 bp)LUC, washed, pooled, and aliquoted into 24-well plates. Cells were pretreated for 30 min with the following antibodies: Ab5 (control IgG), 5 (anti- 5-integrin), anti-CD11b, anti-CD14, and 2 (anti- 2-integrin). Cells were incubated in the presence or absence of LPS (5 µg/ml) for 20 h at 37°C and 5% CO2. Cells were harvested, washed, resuspended in 100 µl of cell lysis buffer, and tested for luciferase activity. Results were recorded as relative luciferase units and standardized for transfection efficiency using -galactosidase activity. Data are presented as means ± SD (n = 9). Note that the anti-CD14 antibody was able to block the LPS induction of the 5 gene transcription (P < 0.001).
|
|
 |
DISCUSSION
|
---|
During an infection with gram-negative bacteria, host immune cells become activated and migrate into affected tissues where they play a major role in the inflammatory response. LPS has been shown to trigger the release of many host inflammatory factors such as chemokines, cytokines, and cell adhesion molecules. Exposure of host cells to infectious organisms or their cell wall components stimulate the production and release of extracellular matrix molecules, such as collagen and fibronectin, from macrophages, epithelial cells, and fibroblasts in the affected area. These events combine to aid in the migration and activation of immune cells that are capable of detecting insoluble gradients created by the differential expression of matrix components at the site of infection (35, 37). The degradation of these extracellular matrix components via an increase in the expression and activation of matrix-degrading proteases (e.g., matrix metalloproteinases) also creates the release of chemotactic fragments for attracting immune cells. Fibronectin fragments have been shown to be chemotactic toward monocytes and neutrophils, among other cells (25, 27). This binding of immune cells to fibronectin or fibronectin fragments occurs through the fibronectin receptor or
5
1-integrin. Binding to the
5
1-integrin receptor then activates a cascade of downstream signal transduction events that in turn lead to the activation of transcription factors, including members of the NF-
B/Rel family that activate inflammatory genes like tumor necrosis factor-
and interleukin-1 (7, 38).
In this report, we explore in detail how LPS stimulates the expression of functional
5
1 receptors. Our studies expand on others, demonstrating increased bacterial adherence to fibronectin and endothelial cells in response to LPS (49). We show that LPS treatment of U937 cells and PBMCs increases cell adhesion to fibronectin, a process likely to aid immune cell tropism into infected tissues. Of note, we find that an antibody against another integrin subunit,
2, also enhanced cell adhesion to fibronectin. This might be related to IgG-induced receptor clustering and induction of an "outside in" or "inside out"
2 receptor signaling that culminates in increased
5
1 function and/or expression. Further studies of the
2-integrin receptor are needed to resolve this issue. In addition, we show that the lipid A component of LPS stimulates the transcription of the gene coding for the
5-integrin subunit that results in accumulation of
5 mRNA and an increased expression of
5
1-integrin receptors. These events are mediated via CD14-dependent signals that include PKC activation.
Many host molecules have been shown to stimulate the expression of integrins. For example, transforming growth factor-
, a product of the tissue remodeling response triggered after injury, is well known for its ability to stimulate the expression of
5
1 and other integrins (34). Phorbol esters are also capable of this response (4). However, less is known about how pathogenic bacteria can promote this response. Bacterial surface molecules like LPS also show agonistic activity toward integrin expression (16). The ability of LPS to stimulate integrin expression differs depending on the integrin and on the immune cell type and differentiation state being studied. For example, in contrast to alveolar macrophages, monocytes show an increase in the expression of CD11a, CD11b, and CD11c and a decrease in L-selectin when exposed to LPS (15). Others have shown the induction of
1
1-integrin in monocytes exposed to LPS and interferon-
(41).
In this report, the LPS-induced integrin response was found to be antagonized by anti-CD14 antibodies. This is not surprising since CD14 receptors are known to mediate many of the effects of LPS, at least indirectly. LPS fails to elicit tumor necrosis factor-
and interleukin-6 production in macrophages obtained from CD14-null mice (24). CD14 is a glycosylphosphatidyl inositol-linked surface protein found on macrophages and other cells (with a soluble form present in the serum) that is responsible for lipid A recognition (11, 31, 50). The NH2-terminal domain of CD14 contains the LPS binding site and is sufficient for cell activation (31). LBP apparently functions as a lipid transfer protein that delivers lipid A to CD14 receptors.
Once bound, CD14 appears to redirect LPS to other transmembrane proteins, such as members of the TLRs like TRL4 (and MD-2 accessory protein) and TLR6 (18). TRL4 signals through myeloid differentiation factor 88 to activate transcription factors NF-
B, ELK-1, and AP-1 (23). Ultimately, LPS induction of these transmembrane proteins generates key intracellular signals that stimulate the production of cytokines, enzymes (such as nitric oxide synthetase), and other non-protein mediators (e.g., platelet-activating factor), many of which are considered important for the development of clinical presentation of endotoxin-induced shock (13, 20, 32). These transcriptional events seem to be related to the rapid activation of specific proteins via tyrosine phosphorylation, specifically mitogen-activated protein kinase (MAPK) and NF-
B (31). Interestingly,
-cells, among other cell types, lack CD14 receptors but nevertheless respond to LPS, suggesting an alternative pathway. It seems that these cells use a soluble CD14 that forms a complex with LPS that is recognized by an unknown component on the cell surface, leading to the release of cytokines and upregulation of adhesion molecules (11, 13, 44, 50, 51). This alternative pathway can be used by CD14-bearing cells in environments where LBP is low (11, 13, 44, 50). This and other observations suggest that CD14 is essential for macrophage responses to free LPS, but that other receptors (e.g., CD11b/CD18) can assist in responding to intact bacteria (39).
Role of PKC and transcription factor activation.
Herein, we demonstrate that LPS induces the expression and activity of cPKC-
and that this signaling molecule is important for LPS-induced integrin expression. In monocytes and macrophages, other signaling pathways activated by LPS include phospholipases A (10) and C (6), other PKC isoforms (43), and the Src family tyrosine kinases Hck, Lyn, and Fgr (46). LPS also induces the phosphorylation of Vav (12) and the activation of the MAPK family p42/p44 (21).
Some of the signaling events described above have been shown to induce, among other things, the expression of monocytic genes by activating the NF-
B/Rel transcription factor family. This was found to be true in our system where induction of NF-
B DNA binding by LPS was found to be important for
5 gene transcription. Others have shown in unstimulated monocytes that NF-
B is retained in the cytoplasm by binding to a family of inhibitors (I
B-
, I
B-
, I
B-
). Because LPS induces I
B kinase activity in human monocytes and THP-1 monocytic cells, it is likely that LPS affects NF-
B activity in more than one way (26).
Implications.
Together, our data suggest that the interaction of host monocytic cells with bacterial-derived LPS triggers intracellular signals that stimulate the expression of
5
1-integrin receptors. In view of many demonstrated functions of
5
1 related to cell migration, chemotaxis, and cellular activation, this might represent a mechanism of tissue tropism by which the host enhances its capability of delivering immune cells into tissues during active infection. However, the unopposed stimulation of these signals might lead to an overwhelming inflammatory response that results in tissue destruction and shock. This is consistent with the idea that it is the host response that initiates endotoxin injury (1, 11, 47). Lipid A, for example, is not directly toxic since cells that lack CD14 can grow in the presence of LPS. Also, transgenic mice that lack CD14 are more resistant to LPS than normal mice (31, 32, 47, 51). Therefore, a better understanding of the mechanisms of LPS recognition and responses may allow us to better identify patients at risk for the development of endotoxin-mediated shock and aid in the development of effective therapies.
 |
GRANTS
|
---|
This work was supported by a Department of Defense grant to J. Roman.
 |
FOOTNOTES
|
---|
Address for reprint requests and other correspondence: J. Roman, Emory Univ., Dept. of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine, Whitehead Biomedical Research Bldg., 615 Michael St., Ste. 205M, Atlanta, GA 30322 (E-mail: jroman{at}emory.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
 |
REFERENCES
|
---|
- Akiyama SK, Yamada SS, Chen WT, and Yamada KM. Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization. J Cell Biol 109: 863875, 1989.[Abstract]
- Alessi DR, Cuenda A, Cohen P, Dudley DT, and Saltiel AR. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase in vitro and in vivo. J Biol Chem 270: 2748927494, 1995.[Abstract/Free Full Text]
- Arnaout MA. Leukocyte adhesion molecules deficiency: its structural basis, pathophysiology, and implications for modulating the inflammatory response. Immunol Rev 114: 145180, 1990.[ISI][Medline]
- Boles BK, Ritzenthaler J, Birkenmeier T, and Roman J. Phorbol ester-induced U937 differentiation: effects on integrin
5 gene transcription. Am J Physiol Lung Cell Mol Physiol 278: L703L712, 2000.[Abstract/Free Full Text]
- Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248254, 1976.[CrossRef][ISI][Medline]
- Buscher D, Hipskind RA, Krautwald S, Reimann T, and Baccarini M. Ras-dependent and -independent pathways target the mitogen-activated protein kinase network in macrophages. Mol Cell Biol 15: 466475, 1995.[Abstract]
- Chang ZL, Beezhold DH, Personius CD, and Shen ZL. Fibronectin cell-binding domain triggered transmembrane signal transduction in human monocytes. J Leukoc Biol 53: 7985, 1993.[Abstract]
- Chomczynski P and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156159, 1987.[CrossRef][ISI][Medline]
- Dignam JD, Lebovitz RM, and Roeder RG. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11: 14751489, 1983.[Abstract]
- Forehand JR, Johnston RB Jr, and Bomalaski JS. Phospholipase A2 activity in human neutrophils. Stimulation by lipopolysaccharide and possible involvement in priming for an enhanced respiratory burst. J Immunol 151: 49184925, 1993.[Abstract/Free Full Text]
- Frey EA, Miller DS, Jahr TG, Sundan A, Bazil V, Espevik T, Finlay BB, and Wright SD. Soluble CD14 participates in the response of cells to lipopolysaccharide. J Exp Med 176: 16651671, 1992.[Abstract]
- Geng Y, Gulbins E, Altman A, and Lotz M. Monocyte deactivation by interleukin 10 via inhibition of tyrosine kinase activity and the Ras signaling pathway. Proc Natl Acad Sci USA 91: 86028606, 1994.[Abstract]
- Glauser MP, Zanetti G, Baumgartner JD, and Cohen J. Septic shock: pathogenesis. Lancet 338: 732736, 1991.[ISI][Medline]
- Graves KL and Roman J. Fibronectin modulates expression of interleukin-1
and its receptor antagonist in human mononuclear cells. Am J Physiol Lung Cell Mol Physiol 271: L61L69, 1996.[Abstract/Free Full Text]
- Haugen TS, Skjonsberg OH, Nakstad B, and Lyberg T. Modulation of adhesion molecule profiles on alveolar macrophages and blood leukocytes. Respiration 66: 528537, 1999.[CrossRef][ISI][Medline]
- Kang YH, Lee CH, Brummel SE, Newball HH, and Forrester J. Effects of endotoxin on expression of VLA integrins by human bronchoalveolar lavage macrophages. J Leukoc Biol 57: 624634, 1995.[Abstract]
- Kobayashi E, Nakano H, Morimoto M, and Tamaoki T. Calphostin C (UCN-1028C), a novel microbial compound, is a highly potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun 159: 548553, 1989.[ISI][Medline]
- Kopp EB and Medzhitov R. The Toll-receptor family and control of innate immunity. Curr Opin Immunol 11: 1318, 1999.[CrossRef][ISI][Medline]
- Landegren UL. Measurements of cell numbers by means of endogenous enzyme hexosaminidase: applications to detection of lymphokines and cell surface antigens. J Immunol Methods 67: 379388, 1984.[CrossRef][ISI][Medline]
- Leturcq DJ, Moriarty AM, Talbott G, Winn RK, Martin TR, and Ulevitch RJ. Antibodies against CD14 protect primates from endotoxin-induced shock. J Clin Invest 98: 15331538, 1996.[Abstract/Free Full Text]
- Liu MK, Herrera-Velit P, Brownsey RW, and Reiner NE. CD14-dependent activation of protein kinase C and mitogen-activated protein kinases (p42 and p44) in human monocytes treated with bacterial lipopolysaccharide. J Immunol 153: 26422652, 1994.[Abstract/Free Full Text]
- Livak KJ and Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-

CT method. Methods 25: 402408, 2001.[CrossRef][ISI][Medline]
- Medvedev AE, Lentschat A, Wahl LM, Golenbock DT, and Vogel SN. Dysregulation of LPS-induced Toll-like receptor 4-MyD88 complex formation and IL-1 receptor-associated kinase 1 activation in endotoxin-tolerant cells. J Immunol 169: 52095216, 2002.[Abstract/Free Full Text]
- Moore KJ, Andersson LP, Ingalls RR, Monks BG, Li R, Arnaout MA, Golenbock DT, and Freeman MW. Divergent response to LPS and bacteria in CD14-deficient murine macrophages. J Immunol 165: 42724280, 2000.[Abstract/Free Full Text]
- Norris DA, Clark RA, Swigart LM, Huff JC, Weston WL, and Howell SE. Fibronectin fragment(s) are chemotactic for human peripheral blood monocytes. J Immunol 129: 16121618, 1982.[Abstract/Free Full Text]
- O'Connell MA, Bennett BL, Mercurio F, Manning AM, and Mackman N. Role of IKK1 and IKK2 in lipopolysaccharide signaling in human monocytic cells. J Biol Chem 273: 3041030414, 1998.[Abstract/Free Full Text]
- Parekh T, Saxena B, Reibman J, Cronstein BN, and Gold LI. Neutrophil chemotaxis in response to TGF-
isoforms (TGF-
1, TGF-
2, TGF-
3) is mediated by fibronectin. J Immunol 152: 24562466, 1994.[Abstract/Free Full Text]
- Perez RL, Rivera-Marrero CA, and Roman J. Pulmonary granulomatous inflammation: from sarcoidosis to tuberculosis. Semin Respir Infect 18: 2332, 2003.[CrossRef][Medline]
- Perez RL, Roman J, Roser S, Little C, Olsen M, Indrigo J, Hunter RL, and Actor JK. Cytokine message and protein expression during lung granuloma formation and resolution induced by the mycobacterial cord factor trehalose-6,6'-dimycolate. J Interferon Cytokine Res 20: 795804, 2000.[CrossRef][ISI][Medline]
- Perez RL, Roman J, Staton GW Jr, and Hunter RL. Extravascular coagulation and fibrinolysis in murine lung inflammation induced by mycobacterial cord factor trehalose-6,6'-dimycolate. Am J Respir Crit Care Med 149: 510518, 1994.[Abstract]
- Pugin J, Shurer-Malay CC, Leturcq D, Moriarty A, Ulevitch RJ, and Tobias PS. Lipopolysaccharide activation of human endothelial and epithelial cells is mediated by lipopolysaccharide-binding protein and soluble CD14. Proc Natl Acad Sci USA 90: 27442748, 1993.[Abstract]
- Raetz CRH. Bacterial lipopolysaccharides: a remarkable family of bioactive macroamphiphiles. In: Escherichia Coli and Salmonella. Cellular and Molecular Biology, edited by Niedhardt FC. Washington, DC: Am Soc Microbiol, 1996, p. 10351057.
- Ritzenthaler J and Roman J. Differential effects of protein kinase C inhibitors on fibronectin-induced interleukin 1-
gene transcription, protein synthesis and secretion in human monocytic cells. Immunology 95: 264271, 1998.[CrossRef][ISI][Medline]
- Roberts CJ, Birkenmeier TM, McQuillan JJ, Akiyama SK, Yamada SS, Chen WT, Yamada KM, and McDonald JA. Transforming growth factor
stimulates the expression of fibronectin and of both subunits of the human fibronectin receptor by cultured human lung fibroblasts. J Biol Chem 263: 45864592, 1988.[Abstract/Free Full Text]
- Roman J. Extracellular matrix and lung inflammation. Immunol Res 15: 163178, 1996.[ISI][Medline]
- Roman J, LaChance RM, Broekelmann TJ, Kennedy CJ, Wayner EA, Carter WG, and McDonald JA. The fibronectin receptor is organized by extracellular matrix fibronectin: implications for oncogenic transformation and for cell recognition of fibronectin matrices. J Cell Biol 108: 25292543, 1989.[Abstract]
- Roman J and McDonald JA. Fibronectins and fibronectin receptors in lung development, injury and repair. In: The Lung: Scientific Foundations (2nd ed.), edited by Crystal RG, West JB, Barnes P, Cherniack N, and Weibel ER. Philadelphia, PA: Lippincott-Raven, 1997, p. 737755.
- Roman J, Ritzenthaler JD, Fenton MJ, Roser S, and Schuyler W. Transcriptional regulation of the human interleukin-1
gene by fibronectin: role of protein kinase C and activator protein-1 (AP-1). Cytokine 12: 15811596, 2000.[CrossRef][ISI][Medline]
- Romer LH and Polin RA. Endotoxin, tumor necrosis factor, and dexamethasone effects on human endothelial cell fibronectin dynamics, synthesis, matrix assembly, and receptor expression. Biochem Cell Biol 73: 515524, 1995.[ISI][Medline]
- Rouslahti E. Fibronectin and its receptors. Annu Rev Biochem 57: 375413, 1988.[CrossRef][ISI][Medline]
- Rubio MA, Sotillos M, Joehems G, Alvarez V, and Corbi AL. Monocyte activation: rapid induction of
1/
1 (VLA 1) integrin expression by lipopolysaccharide and interferon-
. Immunol 25: 27012705, 1995.
- Schumann RR, Leong SR, Flaggs GW, Gray PW, Wright SD, Mathison JC, Tobias PS, and Ulevitch RJ. Structure and function of lipopolysaccharide binding protein. Science 249: 14291431, 1990.[ISI][Medline]
- Shapira L, Takashiba S, Champagne C, Amar S, and Van-Dyke TE. Involvement of protein kinase C and protein tyrosine kinase in lipopolysaccharide-induced TNF-
and IL-1
production by human monocytes. J Immunol 153: 18181824, 1994.[Abstract/Free Full Text]
- Snyderman R and Goetzl EJ. Molecular and cellular mechanisms of leukocyte chemotaxis. Science 213: 830837, 1981.[ISI][Medline]
- St-Denis A, Chano F, Tremblay P, St-Pierre Y, and Descoteaux A. Protein kinase C-
modulates lipopolysaccharide-induced functions in a murine macrophage cell line. J Biol Chem 273: 3278732792, 1998.[Abstract/Free Full Text]
- Stefanova I, Corcoran ML, Horak EM, Wahl LM, Bolen JB, and Horak ID. Lipopolysaccharide induces activation of CD14-associated protein tyrosine kinase p53/56lyn. J Biol Chem 268: 2072520728, 1993.[Abstract/Free Full Text]
- Tapping RI and Tobias PS. Cellular binding of soluble CD14 requires lipopolysaccharide (LPS) and LPS-binding protein. J Biol Chem 272: 2315723164, 1997.[Abstract/Free Full Text]
- Tobias PS, Mathison J, Mintz D, Lee JD, Kravchenko V, Kato K, Pugin J, and Ulevitch RJ. Participation of lipopolysaccharide-binding protein in lipopolysaccharide-dependent macrophage activation. Am J Respir Cell Mol Biol 7: 239245, 1992.[ISI][Medline]
- Vercellotti GM, Lussenhop D, Peterson PK, Furcht LT, McCarthy JB, Jacob HS, and Moldow CF. Bacterial adherence to fibronectin and endothelial cells: a possible mechanism for bacterial tissue tropism. J Lab Clin Med 103: 3443, 1984.[ISI][Medline]
- Wright SD, Ramos RA, Tobias PS, Ulevitch RJ, and Mathison JC. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 249: 14311433, 1990.[ISI][Medline]
- Yu B, Hailman E, and Wright SD. Lipopolysaccharide binding protein and soluble CD14 catalyze exchange of phospholipids. J Clin Invest 99: 315324, 1997.[Abstract/Free Full Text]
Copyright © 2004 by the American Physiological Society.