Published online before print May 2, 2008
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* Molmed S.p.A., Milan, Italy; and
Cancer Immunotherapy and Gene Therapy Program, H. San Raffaele Scientific Institute and Università Vita-Salute, Milano, Italy
1Correspondence: Molmed S.p.A., via Olgettina 58, 20132 Milan, Italy. E-mail: barbara.valentinis{at}molmed.com
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B-
/NF-B and ERK1/2 pathways in human DCs. No activation of p38 was induced in the same experimental conditions. Our data suggest that the IB-/NF-B pathway has a critical role in the partial maturation of DCs induced by rHSP70.
Key Words: ERK1/2 • IB-/NF-B • partial maturation • humoral immunity
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Regarding the mechanism through which microbial or mammalian HSP70s exert their effects on APCs, several receptors have been identified: CD91 [20
], CD40 [21
, 22
], TLR-2, and TLR-4 [23
, 24
]. The signal transduction pathways activated by HSP70 of different origin have also been studied. HSP70 activates two of the main pathways activated by LPS and other stimulatory factors [25
26
27
28
]: the IB-/NF-B pathway [7
, 24
] and the p38 stress-activated protein kinase (p38) [22
, 29
]. With the only exception of the report by Wang and co-workers [29
], which investigated the signaling induced by a microbial HSP70 in human DCs, all of the other studies were conducted on human monocytes [7
] or cell lines [22
, 24
, 29
].
The aim of our work was to analyze the activation of signal transduction pathways in human DCs stimulated with a human recombinant HSP70 (rHSP70) purified from eukariotic cells. Moreover, we investigated the adjuvant properties of HSP70 in stimulating the production of antibodies against a soluble protein antigen in vivo. In this report, we show that human HSP70, which induces a partial maturation of human DCs in vitro and elicits an effective adjuvant activity in vivo, selectively activates IB-/NF-B and ERK1/2 pathways in human DCs.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cloning of human rHSP70 and cell line retroviral transduction
The human full-length, inducible HSP70 (ref. [30
] and Gen Bank sequence NM005345) was cloned by RT-PCR from PHA-stimulated human lymphocytes into the pcDNA 4/HisMax vector (Invitrogen, Carlsbad, CA, USA). This vector allowed for the insertion of a poly-histidine (His; 6XHis) tag at the N-terminal of HSP70. We used retroviral vectors for stable expression of the recombinant protein in mammalian cells. The HSP70 was cloned into a murine leukemia virus (MFG)-based vector that allows the transfer of two genes under the transcriptional control of the viral long-terminal repeat. The translation of the second gene in the polycistronic mRNA is allowed by an internal ribosome entry-site sequence. In our construct, we used a mutated, biologically inactive form of the low-affinity nerve growth factor receptor (
LNGFr; ref. [31
]) as a marker gene, which allows detection and isolation of the transduced cells. NIH3T3 cells were transduced for 4 h in the presence of polybrene (4 µg/ml), and retroviral stocks were generated with a transient expression system. Five days later, cells were analyzed for the expression of the LNGFr at the flow cytometer, and LNGFr-positive cells were sorted by immunomagnetic beads (Dynabeads, Dynal, Norway).
Purification of human rHSP70
NIH3T3 cells expressing rHSP70 were lysed in 50 mM Tris, pH 8.0, 50 mM NaCl, 0.5% Nonidet P-40 buffer containing protease inhibitors. The human rHSP70 was purified by nondenaturing affinity chromatography using prepacked, Ni2+-charged chelating Sepharose columns, following the manufacturer’s instruction (Amersham Biosciences, GE Healthcare, UK) and ultra-filtered on polyetersulfone membranes with a 30,000 m.w. cut-off (Vivascience, Germany). Immunoblots with anti-His or anti-HSP70 antibodies were performed to check for the identity of the protein. Purity was evaluated by Coomassie blue staining (BioRad, Hercules, CA, USA) of a SDS-PAGE gel, in which 1 µg rHSP70 was run. The purified protein was tested for endotoxin contamination by the kinetic-QLC Limulus amoebocyte lysate (LAL) cell test. The content of the endotoxin assessed was under the limits of detection (<0.06 EU/ml) for a concentration of rHSP70 up to 50 µg/ml.
Generation of monocyte-derived DCs
Human DCs were generated from the adherent fraction of PBMC of healthy donors in the presence of GM-CSF and IL-4, as described previously [32
]. At Day 5 of culture, only detached, floating cells were collected and used in experiments as immature DCs. These cells are in fact fully differentiated DCs, as shown by their phenotype (CD14-negative and CD1a-positive).
Quantification of cell adhesion and flow cytometric analysis
Immature DCs were seeded at 105 cells/cm2 in 0.5 ml RPMI 1640 supplemented with 10% FBS, 800 units/ml GM-CSF, and 100 units/ml IL-4. As described previously [32
], to quantify cell adhesion, cells in suspension were collected and counted by trypan blue exclusion separately from adherent cells, which were detached by incubation in PBS with 2 mM EDTA. The analysis of cell-surface antigens was performed at 48 h on the whole population of suspended and adherent cells, which were incubated with the anti-human CD86-FITC-conjugated antibodies (PharMingen, BD Biosciences, San Diego, CA, USA) for 30 min on ice. HLA class I was detected by indirect immunofluorescence using the specific mAb W6/32 for 30 min on ice. Appropriate isotype controls (PharMingen, BD Biosciences) were used. Samples were analyzed using the Beckman-Coulter flow cytometer (Beckman-Coulter, Fullerton, CA, USA).
Detection of cytokines and T lymphocyte activation assay
Supernatants of DCs were collected at the indicated time-points and subjected to cytokine analysis. IL-10 and IL-12 were evaluated by ELISA, according to the manufacturer’s instruction, using couples of antibodies from PharMingen, BD Biosciences, and Endogen (Woburn, MA, USA), respectively. TNF- released by DCs in the supernatant was determined by testing its cytotoxic effect on WEHI-164.13 in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay [32
]. Lymphocyte activation was evaluated as IFN-
released in the culture medium: DCs (1x105 cells/well) and a MelanA/MART1-specific CD8+ T cell clone (1x104 cells/well) were cocultured (final vol, 100 µl), and the medium was collected after 24 h. IFN- was evaluated by ELISA according to the manufacturer’s instruction (PharMingen, BD Biosciences).
Immunoblots
As described previously [32
], cells in suspension were collected at the indicated time-points, washed in ice-cold PBS, centrifuged, and lysed along with the corresponding PBS-washed, adherent cells. Equal amounts of total proteins were subjected to Western immunoblotting. ERK1/2 and p38 activities or IB- status were evaluated by immunoblotting using antibodies directed against specific phosphorylated amino acids, present only in the active form of the proteins, according to the manufacturer’s instruction (New England Biolabs, Beverly, MA, USA). We used an antiphospho-ERK1/2 (Thr202/Tyr204) mAb, an antiphospho-p38 MAPK (Thr180/Tyr182) polyclonal antibody, and an antiphospho-IB- (Ser32) polyclonal antibody. Anti-ERK1/2, -p38, -IB- (New England Biolabs), and -actin (Sigma-Aldrich) polyclonal antibodies were used to evaluate the amount of total proteins present on the membrane.
In vivo immunization
Three strains of mice were used: C57BL/6 mice; wild-type, LPS-responsive C3H/HeN mice; and C3H/HeJ mice, which are hyporesponsive to LPS as a result of a mutation in TLR-4 (Jackson Laboratory, Bar Harbor, ME, USA). Groups of five mice were injected s.c. twice at Days 0 and 14, with purified OVA (10 µg/mouse, Sigma-Aldrich), alone or in combination with rHSP70 (5 µg/mouse), CFA (Pierce, Rockford, IL, USA), or LPS (50 µg/mouse). rHSP70 was mixed to albumin just before the injection. As a control, cohorts of mice were injected with PBS or rHSP70 alone. At Day 24, OVA-specific antibodies were assessed in triplicate by ELISA: 96-well plates (Nunc) were coated with OVA (10 µg/ml) and blocked with 20% FCS; diluted sera were added, and samples were developed by sequential incubation with HRP-conjugated, IgG-specific mAb and substrate (Sigma-Aldrich). Results are expressed as end-point titers, which are the highest serum dilution that gave an OD reading higher then 0.5. Anti-OVA Ig isotyping was performed by sequential incubation with specific anti-mouse IgG isotype mAb (PharMingen, BD Biosciences); HRP-conjugated, anti-rat, mouse-adsorbed antibodies; and substrate (Sigma-Aldrich). Results are expressed as OD values minus the corresponding background.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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LNGFr [31
], a cell surface marker used to monitor transduction efficiency and to select pure populations of transduced cells. NIH3T3 cells were transduced, selected, and controlled for mycoplasm contamination. We purified human-inducible rHSP70 from cell lysates of transduced NIH3T3 cells by affinity chromatography. Total lysates were diluted in a phosphate buffer containing 40 mM imidazole before chromatography. As shown in Figure 1A
and 1B
, the retained rHSP70 was eluted from the column by 150 mM imidazole. Fractions containing the rHSP70 were pooled and ultra-filtered to remove the imidazole and to concentrate the rHSP70 in Dulbecco’s PBS. The purified protein was characterized by immunoblotting with anti-HSP70 or anti-His antibodies (Fig. 1C)
. In both cases, only a single band of the expected size was detected. Purity was confirmed by Coomassie blue staining of a SDS-PAGE gel, in which 1 µg of the protein was run (Fig. 1C)
.
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Figure 1. Expression and purification of human rHSP70. (A and B) HSP70 was purified from NIH3T3 cells expressing human rHSP70 by affinity chromatography using a Ni2+-charged chelating Sepharose column. Total lysate (TL) was diluted with a phosphate buffer containing 40 mM imidazole and applied to the column. A negligible amount of rHSP70 was lost in the flow-through (FT). After an extensive wash with 40 mM imidazole phosphate buffer (WI and WII), rHSP70 was eluted by increasing the imidazole concentration to 150 mM. The purification process was monitored on aliquots of the chromatographic fractions by SDS polyacrylamide gel followed by Coomassie blue staining (A) or immunoblotting with specific antibodies (B). (C) Recombinant protein (1 µg) was run on a SDS polyacrylamide gel and immunoblotted with the indicated antibodies (left) or stained with Coomassie blue (right).
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The use of the LPS-inactivating reagent Polymyxin B in our experiments was not feasible because of its effects on cellular transcription [34
] and its ability to induce partial maturation in human DCs [32
]. Therefore, we set up several experiments to exclude a possible interference of contaminating LPS in our system. The HSP70 lots were tested for endotoxin contamination by the kinetic-QLC LAL cell test. The content of endotoxin assessed was under the limit of detection (<0.06 EU/ml) for a concentration of HSP70 up to 50 µg/ml. As the protein was used at a final concentration lower than 50 µg/ml, the possible effect of contaminating endotoxin is ruled out. Moreover, we performed further in vitro and in vivo experiments aimed to demonstrate the absence of endotoxin contaminations. It has been reported by Thurnher and collaborators [35
] that incubation of human monocytes with low doses of LPS during their differentiation in DCs inhibits the release of cytokines from DCs upon a subsequent stimulation with high doses of LPS. Similarly, we found that immature DCs incubated for 24 h with LPS (0.1 ng/ml) were desensitized to a subsequent challenge with 100 ng/ml LPS (Fig. 2
). We observed a significant decrease (P<0.001) in the amount of TNF-, IL-10, and IL-12 released at 48 h (24 h of preincubation plus 24 h of treatment) by DCs preincubated with LPS at 0.1 ng/ml. We repeated the experiment preincubating DCs with rHSP70 (30 µg/ml), and we found that rHSP70 did not desensitize DCs to a subsequent challenge with LPS (Fig. 2)
. This result confirms that rHSP70 used in our experiments did not contain LPS contamination.
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Figure 2. HSP70 does not desensitize DC maturation induced by LPS. (A–C) Human immature DCs were pretreated for 24 h with rHSP70 (30 µg/ml) or LPS (0.1 ng/ml; LPS/L) or left untreated (–). Then, DCs were stimulated by adding rHSP70 (30 µg/ml), LPS (0.1 ng/ml; LPS/L), LPS 100 ng/ml (LPS/H), or PBS as control (–) for an additional 24 h. The supernatants (conditioned by overall 48 h) were tested for cytokine content. Detection of TNF- (A), IL-10 (B), or IL-12/p70 (C) in the medium was performed as described in Materials and Methods. The values are mean of triplicate determinations with SD. A representative experiment of three is shown. *, The value compared with –/LPS/H is statistically significant, P < 0.001.
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, IL-10, and IL-12 (Fig. 2A
2B
2C)
but up-regulated the expression of HLA class I molecules (data not shown) and of the CD86 costimulatory molecule (Fig. 3B)
. The up-regulation observed was lower compared with that induced by optimal concentration of LPS but consistent and reproducible (P<0.05), and it was obtained with three different batches of HSP70.
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Figure 3. In vitro maturation and activation of rHSP70-treated human DCs. (A) Immature human DCs were incubated in the presence of rHSP70 (30 µg/ml) or LPS (100 ng/ml) for 4 h. In some samples, rHSP70 and LPS were pretreated at 99°C for 30 min (rHSP70/D and LPS/D, respectively). Cells in suspension (black bars) or cells adherent to the plate (gray bars) were collected and counted as described in Materials and Methods. The white bars are the total number of cells obtained, counting suspended and adherent cells for each condition. The percentage of adherent cells on the total number of cells is reported for each experimental condition. A representative experiment (mean with SD) of four independent experiments is shown. *, Statistically significant values, P < 0.05. (B) Immature DCs were cultured for 48 h alone (untreated) in the presence of rHSP70 (30 µg/ml) or LPS (100 ng/ml), and CD86 expression was evaluated by flow cytometry as percent of positive cells. Results are depicted as mean ± SE of three independent experiments performed with DCs from different donors (*, statistically significant values, P<0.05). (C) A CD8+ T cell clone (CTL) was cocultured with DCs in the presence of rHSP70 (3 µg/ml). In some samples, rHSP70 was pretreated at 99°C for 30 min (rHSP70/D). The IFN- released by the CTL in the medium was determined by ELISA after 24 h of incubation. The effect of rHSP70 on the DCs or CTL alone was also evaluated. Results are expressed as mean ± SE of three independent experiments. *, Statistically significant values, P < 0.05.
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by the T cell clone (Fig. 3C)
. Heat-denatured rHSP70 did not influence the ability of DCs to activate T cells anymore in a significant way (P<0.05), further confirming that conformational integrity of the protein is required. These results suggest that rHSP70 induces a functional, partial maturation of DCs in vitro.
HSP70 activity in vivo
We then verified whether rHSP70 had an adjuvant effect in vivo by testing its ability to modulate the primary antibody response against an exogenous soluble antigen. We immunized wild-type C57BL/6 mice by injecting OVA s.c. in the presence or in the absence of rHSP70. In mice treated with rHSP70 and OVA (combined just before the injection), we observed an increase in OVA-specific IgG antibody titers with respect to OVA-treated mice (Fig. 4A
). The IgG titer observed in mice immunized with OVA plus rHSP70 was lower than the titer in serum from mice immunized with OVA plus CFA (end-point dilution greater than 6x102; data not shown), an oil-in-water emulsion containing heat-killed Mycobacterium tuberculosis that is the gold standard adjuvant for immunization in experimental animals. No specific antibodies were detected in control cohorts of mice injected with PBS or rHSP70 alone (data not shown). Similar adjuvant effects of rHSP70 on the induction of the anti-OVA humoral response were obtained in wild-type, LPS-responsive C3H/HeN as well as in C3H/HeJ mice, which are LPS-ipo-responsive as a result of a mutation in TLR-4 (Fig. 4A)
. As we obtained a similar response in the three strains of mice (C57BL6, C3H/HeN, and C3H/HeJ), we therefore compared all OVA + HSP70-treated mice (15 animals) with mice treated with OVA alone (15 mice). As shown in Figure 4A
, the increase in OVA-specific IgG antibody titer induced by rHSP70 plus OVA was statistically different from that one induced by OVA alone (P<0.05). As expected, a remarkable decrease in the antibody titer was observed in LPS ipo-responsive mice treated with OVA plus LPS (data not shown). These results indicate that rHSP70 has an adjuvant activity in vivo also with soluble antigen and that its effect is not a result of a LPS contamination.
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Figure 4. In vivo adjuvant activity of rHSP70 toward soluble antigen. (A) The production of OVA-specific IgG was evaluated by ELISA in serum of C57BL/6, C3H/HeN, or C3H/HeJ mice (five animals for each group of treatment), vaccinated with OVA alone (white bars, 10 µg/mouse) or in combination with rHSP70 (gray bars, 5 µg/mouse). The group named Overall includes all of the 15 mice of the three strains (C57BL/6, C3H/HeN, and C3H/HeJ). Results are expressed as mean ± SE of IgG end-point dilution. *, Statistically significant values, P < 0.05. (B) The anti-OVA-specific Ig isotyping was performed in duplicate on the serum of three C3H/HeN mice for each treatment. The isotypes quantified were IgG1 (white), IgG2b (light gray), IgG2a (dark gray), and IgG3 (black). Results are expressed as mean of the OD reading at the serum dilution 1:20. Identical results were obtained at the dilution 1:40.
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rHSP70 signaling in DC maturation
To investigate the signaling of rHSP70, we analyzed the activation of IB-/NF-B, ERK1/2, and p38 pathways, which have been shown to be involved in the maturation of human DCs by several stimuli [25
26
27
28
]. rHSP70 induced the phosphorylation of IB- at short-term (Fig. 5A
) and of ERK1/2 at short- and long-term (Fig. 5A
and 5B)
. In contrast, no activation of p38 by rHSP70 was observed at either time-points (Fig. 5A
and 5B)
. The intensity of IB-/NF-B and ERK1/2 pathway activation by rHSP70 was lower with respect to the LPS-induced activation but comparable with that induced by "weak", maturative stimuli such as polymyxin B [32
].
 View larger version (22K): [in a new window] |
Figure 5. Signal transduction pathways activated by rHSP70 in human DCs. (A and B) Human DCs treated with rHSP70 (30 µg/ml) or LPS (100 ng/ml) were lysed at short times (A, min) or at 48 h (B). Lysates were run by SDS-PAGE, transferred into a nitrocellulose membrane, and blotted with the indicated antiphospho antibodies. After stripping, each membrane was reblotted with a specific antibody to monitor for the total level of protein loaded (anti-ERK1/2, anti-p38, anti-IB-, and anti-actin). Shown is one out of three independently performed experiments.
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B-/NF-B pathway, partially inhibited the CD86 and HLA class I expression induced by HSP70 (data not shown). On the contrary, no effects were observed by incubation with the ERK1/2 inhibitor PD98059 (data not shown).
These results indicate that rHSP70 activates in human DCs the IB-/NF-B and ERK1/2 pathways and suggest that IB-/NF-B is required for DC partial maturation.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, we characterized the signaling events that are downstream of the action of the mammalian HSP70 on primary human DCs, the most potent APCs. To this aim, we expressed the human sequence of HSP70 in eukaryotic cells to reduce the risk of microbial contaminations and to obtain a pattern of post-translational modifications as close as possible to the physiological conditions.
We found that human rHSP70 purified from eukaryotic cells induces in primary human DCs an activation of IB-/NF-B and ERK1/2 pathways with no p38 activation. The IB-/NF-B activation induced by rHSP70 seems to be required for the induction of a partial DC maturation. On the contrary, the role of ERK1/2 activation remains to be elucidated. An involvement of the NF-B pathway in the action of HSP70 on human monocytes has been reported previously [7
]; apparently, various human APCs, therefore, rely on the same intracellular signaling pathway in response to HSP70. Interestingly, Wang et al. [29
] reported that p38 is activated in human DCs by a microbial HSP70 produced in prokaryotes. These data indicate that differences in the sequences or in the post-translational modifications between microbial and mammalian proteins may indeed play a critical role. To our knowledge, the ability of HSP70 to activate the ERK1/2 pathway has never been investigated.
Candidate receptors for the action of rHSP70 include TLR-4 [24 ]. Our results suggest that TLR-4 is not responsible for the observed effects; in fact, rHSP70 was still active in TLR-4 ipo-sensitive mice. Further studies are warranted to verify whether other molecules and in particular, CD40 [21 , 22 ] are actually involved.
Functionally, rHSP70 up-regulates the expression of HLA class I, the costimulatory CD86 molecule, and induces an increased adhesion of primary human DCs. Modulation of adhesion molecules and motility properties in DCs during maturation has been described extensively for LPS and other maturative stimuli [32 , 37 38 39 40 ]. For HSPs, it has only been reported that the expression of adhesion molecules is up-regulated by bacterial HSPs in human monocytes [41 ] and by Chlamydial and human HSP60 in human macrophages [42 ].
The partial maturation and the signaling induced by rHSP70 on human DCs in vitro closely resemble the effects induced by polymyxin B, an antibiotic widely used for its property of binding and inactivating LPS [32
]. Therefore, we propose that adhesion and IB-/NF-B activation have a direct role in the partial maturation induced in primary human DCs by physiological (HSP70) and artificial (polymyxin B) weak stimuli. The maturation promoted by these stimuli is only partial (up-regulation of only CD86 and HLA-class I molecules and no induction of cytokines secretion), probably because of a limited NF-B activation, lower compared with that induced by optimal stimuli such as LPS, which in turn promote DC full maturation. In agreement, NF-B has been described recently as a master regulator of early and late gene expression in primary DCs stimulated by LPS [43
]. Moreover, it has been reported that putative NF-B sites are present in murine and human CD86 promoters [44
].
Until now, in vivo studies have been performed with rHSP70 fused to a protein fragment or with HSP70 complexed naturally or in vitro with peptides [13 14 15 16 17 18 ]. Only recently, it has been shown that HSP70 acts as adjuvant for unlinked peptides by inducing T cell-mediated responses [19 ]. Our report extends this observation to a model protein antigen (i.e., OVA) and to the humoral branch of the immune system. In our experimental system, rHSP70 elicits a partial in vitro maturation of DCs but exerts a significant adjuvant activity in vivo. These results are in agreement with the study of Millar et al. [19 ], where spleen cells or bone marrow-derived DCs did not up-regulate costimulatory molecules after treatment with rHSP70 but were functional in vivo.
The degree of the humoral response induced by rHSP70 is lower and qualitatively different from that induced by LPS or CFA. Indeed, the IgG isotype pattern induced by rHSP70 is predominantly of Th2-type (i.e., IgG1 and IgG2b>IgG2a and IgG3), and that elicited by LPS is Th1-oriented (i.e., IgG2a and IgG3).
Altogether, our results fit the model recently proposed by Randolph and collaborators [45 ] about the strength and duration of signaling in DCs: weak maturation signals preferentially lead to the activation of ERK1/2 with respect to p38, inducing a partial maturation that may be sufficient to sustain Th2-oriented immunity. Conversely, strong maturation stimuli such as LPS, through p38 activation and a complete DC maturation, trigger Th1 polarization [46 ].
We cannot exclude that other molecules may synergize in vivo to increase the adjuvant activity of HSPs in the induction of humoral immune responses or that rHSP70 acts in vivo directly on other immune cells, in particular, B cells. Indeed, it has been reported that HSP70 from Leishmania infantum or Toxoplasma gondii induces proliferation of B lymphocytes [47 , 48 ] and that M. tuberculosis HSP70 enhances class I MHC cross-processing and presentation by B lymphocytes [49 ]. We also found that rHSP70 induced an increase in human B cell proliferation, very modest but similar to that induced by LPS (data not shown).
In conclusion, in this report, we show that a purified human rHSP70, which induces a partial maturation of human DCs in vitro and elicits an adjuvant activity in vivo, activates IB-/NF-B and ERK1/2 pathways in human DCs.
Received August 16, 2007; revised March 10, 2008; accepted March 26, 2008.
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B pathway Int. Immunol. 12,1539-1546
/ BALB/c mice Infect. Immun. 66,347-352B signal transduction pathways are involved in the survival and maturation of lipopolysaccharide-stimulated human monocyte-derived dendritic cells Blood 96,1039-1046, and contact sensitizers J. Immunol. 166,3837-3845B-/NF-B and ERK1/2 pathways and adhesion J. Biol. Chem. 280,14264-14271 and prostaglandin E2 Blood 91,3112-3117B kinase complex and NF-B act as master regulators of lipopolysaccharide-induced gene expression and control subordinate activation of AP-1 Mol. Cell. Biol. 24,6488-6500This article has been cited by other articles:
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