Distinct responses of monocytes to Toll-like receptor ligands and inflammatory cytokines

Cinthia Farina1, Diethilde Theil2, Barbara Semlinger1, Reinhard Hohlfeld1,3 and Edgar Meinl1,3

1 Department of Neuroimmunology, Max-Planck-Institute of Neurobiology, D-82152 Martinsried, Germany, 2 Department of Neurologyand 3 Institute for Clinical Neuroimmunology, Ludwig-Maximilians University, D-81377 Munich, Germany

Correspondence to: E. Meinl; E-mail: meinl{at}neuro.mpg.de
Transmitting editor: G. Hammerling


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study we compared the activation of monocytes by different bacterial products via Toll-like receptors (TLR), and by different proinflammatory mediators. In response to TLR-2, -4 and -5 engagement, ~50% of monocytes produced TNF-{alpha}, compared to only 5% after induction with IFN-{gamma} or GM-CSF. Furthermore, a small proportion of monocytes produced IL-10 after stimulation via TLR, but not after stimulation with cytokines. Both TLR-ligands and inflammatory cytokines induced the expression of CD25, CD69, CD80 and, surprisingly, also of CD83, commonly regarded as an activation marker for mature dendritic cells (DC). Conversely, TLR-ligands downregulated CD38, CD86 and ICOS-L. Importantly, signaling lymphocytic activation molecule (SLAM; CD150) was identified as a monocyte activation marker that could be induced ex novo via TLR-2, -4 and -5, but not by single stimulation with monocyte activators like IL-1, TNF-{alpha}, IFN-ß, IFN-{gamma}, GM-CSF or CD40-L. SLAM expression was transient and required mitogen activated protein kinase (MAPK) p38, but not ERK or JNK, and was surprisingly independent of NF-{kappa}B. SLAM+ monocytes, which are absent in blood, were detected in spleen and tonsils, where they could be localized to T-cell areas and germinal centers. Together, by comparing the response of monocytes to TLR-ligands and inflammatory cytokines, we have identified a monocyte activation marker, SLAM, which differs in its inducibility from other monocyte activation markers. SLAM+ monocytes and macrophages were identified for the first time in vivo. Their presence might be a sign of innate immune activation.

Keywords: cellular activation, inflammation, innate immunity, MAP kinase, SLAM


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The innate immune system represents the first line of defense against microorganisms for it promptly initiates a local inflammatory reaction. The recognition of different pathogen components is mediated by Toll-like receptors (TLRs), which recognize the microbial non-self and mediate activation of the innate immune system (1). On the basis of their homology to the Drosophila Toll protein, 10 TLRs have been described in man so far and the specific ligands of some have been defined (2). TLR-2 binds bacterial lipoproteins and peptidoglican, and TLR-3, -4, -5, -7 and -9 recognize double-stranded RNA, lipopolysaccharides, bacterial flagellin, imiquimod and bacterial DNA, respectively.

TLR engagement induces a variety of responses including cytokine production and expression of costimulatory molecules. This not only kills pathogens, but also activates the adaptive arm of the immune system. The adaptive immune system expands antigen-specific T and B cells in the lymphoid organs and provides long-lasting protection from pathogens by generating memory T and B cells. The magnitude and cytokine polarization of the resulting adaptive immune response is regulated by costimulatory molecules, such as the B7 family and the signaling lymphocytic activation molecule (SLAM; CD150) (3).

Monocytes are essential effector cells in chronic inflammatory diseases and also in fighting infectious agents. To perform their functions, monocytes need to be activated, either via inflammatory cytokines produced by the adaptive immune system or via direct stimulation by bacterial products.

The aim of this study was to elucidate monocyte behavior in vitro after stimulation with bacterial products in comparison with other inflammation mediators. Quantitative analysis of cytokine production and study of the modulation of several surface markers revealed differential monocyte responses. Thereby we identified one monocyte activation marker, SLAM, which was induced by TLR-ligation, but not by single stimulation with inflammatory cytokines. Additional experiments showed that induction of SLAM expression was mediated via the p38 mitogen activated protein kinase (MAPK) in an NF-{kappa}B independent fashion.

SLAM belongs to a family of CD2-related molecules (4). It is expressed on T cells (5), on activated B cells (6), on monocyte-derived mature dendritic cells (79), and on monocytes upon activation with LPS (10,11). In T cells, SLAM costimulation enhances antigen-specific proliferation, the release of IFN-{gamma} (5), and also promotes TCR-mediated cytotoxicity (12). SLAM mediates proliferation and immunoglobulin synthesis in activated B cells (6), as well as IL-8 and IL-12 secretion by mature dendritic cells (8). Moreover, it can serve as alternative receptor for measles virus (13). Since SLAM is a self-ligand (6,14) and expressed by a subset of T cells and B cells, TLR-mediated SLAM induction on monocytes links innate and adaptive immunity. So far SLAM+ monocytes had only been described in vitro, but not in vivo. Therefore we searched for SLAM expression on monocytes in lymphatic tissues. We found SLAM+ monocytes in spleen and tonsils, where they could be localized to T cell areas and germinal centers.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Reagents and antibodies
The following reagents were used: 1000 U/ml human IFN-{gamma}, 25 ng/ml TNF-{alpha} (Roche, Mannheim, Germany), 100 ng/ml IL-1ß, 500 ng/ml IL-2, 100 ng/ml IL-5, 500 ng/ml IL-17, 10 ng/ml GM-CSF, 100 ng/ml IL-12 (R&D, Wiesbaden-Nordenstadt, Germany), 1000 U/ml IL-4 (Promokine, Heidelberg, Germany), 100 ng/ml IL-7 (Peprotech, London, UK), 1000 U/ml IFN-ß (Betaferon, Schering, Berlin, Germany), 5 µg/ml flagellin from Helicobacter pylori (IBT, Reutlingen, Germany), 5 µg/ml peptidoglycan (PGN) from Staphylococcus aureus (Fluka, Sigma-Aldrich, Schnelldorf, Germany), 5 µg/ml lipoteichoic acid (LTA) from Staphylococcus aureus, 300 ng/ml LPS from Escherichia coli 0111:B4 (Sigma-Aldrich), 100 µM Tyr65, Phe67-C5a (65–74) and Trp63, Trp64-C3a (63–77) (Bachem, Heidelberg, Germany), 5 µM CpG ODN (5'-atcgactctcgagcgttctc-3', phosphorothioate backbone, HPSF-purified, from MWH Biotech, Ebersberg, Germany), 25 µg/ml Poly (I:C) (Amersham, Freiburg, Germany), 10 µg/ml recombinant human heat shock protein (HSP)60 and HSP70 (low-endotoxin preparations, <5 pg endotoxin/µg protein), 5 µg/ml purified bovine {alpha}B-crystallin, 5 µg/ml Mycobacterium bovis BCG HSP65 (Biomol, Hamburg, Germany), CD40-ligand kit (2 µg/ml recombinant human CD40L-FLAG, 1 µg/ml crosslinking anti-FLAG mAb, Alexis, Grünberg, Germany), 10 µg/ml anti-human CD40 (BD, Heidelberg, Germany), 10 µM herbimycin A, 10 µM PD98059, 10 µM SB203580, 10 µM U0126, (Calbiochem, Schwalbach, Germany), 10 µM PDTC, 10 µM dexamethasone (Sigma). LPS free reagents, water (BioWhittaker, Verviers, Belgium), PBS (Gibco, Karlsruhe, Germany) and BSA (Sigma) were used to prepare the aliquots. The following antibodies were used for the FACS analysis: PE-labeled anti-SLAM, CD14, CD38, CD83, Per-CP labeled anti-human CD14, FITC labeled anti-human CD25, CD69, TNF-{alpha} (all from BD), PE-labeled anti-human CD86 (Serotec, Eching, Germany), FITC labeled anti-human CD83, pure anti-human CD80 (Immunotech, Dianova, Hamburg, Germany), pure anti-human ICOS-L (clone HIL 131, kindly provided by Dr R. Kroczek) (15), FITC-labeled anti-mouse IgG+M (H+L) (Jackson Laboratories, Dianova, Hamburg, Germany). All the corresponding isotype controls were from BD.

Limulus assay
The endotoxin content in the reagents was measured with the Limulus assay (BioWhittaker): 0.3 pg endotoxin/µg flagellin, 0.06 pg endotoxin/µg PGN, 12 pg endotoxin/µg LTA. We noted that at least some batches of commercially available reagents, including poly (I:C) (Sigma) and heparane sulfate (Seikagaku, Tokyo, Japan) contained amounts of LPS that might have had effects on monocyte activation assays.

Blood samples and cell preparations
Blood was drawn from healthy individuals after their informed consent and PBMC were isolated on a discontinuous density gradient (Lymphoprep, Nycomed, Oslo, Norway). Viable cells were counted by Trypan Blue (Sigma-Aldrich) exclusion and resuspended in culture medium (RPMI 1640 supplemented with 5% FCS, 1% glutamine and 1% penicillin/streptomycin; Gibco). One batch of FCS was used throughout the study. Unstimulated cells were always seeded in parallel.

CD14-enriched cell populations were prepared from PBMC by positive selection with immunomagnetic beads coated with anti-CD14 antibodies from Dynal (Hamburg, Germany) for the Elispot assay experiments or from Miltenyi Biotec (Bergisch Gladbach, Germany) for the FACS experiments. FACS analysis showed that the selected cell populations contained >90% monocytes.

FACS staining and analysis
Briefly, 2 x 105 cells were labeled with the predetermined appropriate antibody dilution or with the corresponding isotype controls for the staining of extracellular markers. For the intracellular FACS staining, PBMC were stimulated in the presence of 10 µg/ml Brefeldin A (Sigma) for 4 h at 37°C, 5% CO2. Cells were then fixed, permeabilized and labeled with anti-TNF-{alpha} antibody or isotype control according to the manufacturer’s protocol. FACS stainings were analyzed on a FACScan using Cell-Quest software (BD). Monocytes were gated in forward/side scatter. The quadrants were set on the relative isotype controls. The experiments were repeated at least three times.

Elispot assay
96-well polyvinylidene difluoride plates (Millipore, Eschborn, Germany) were coated at 4°C overnight with 10 µg/ml capture antibody (anti-TNF-{alpha} Ab or anti-IL-10 Ab; Mabtech, Nacka, Sweden). The plates were then washed and blocked with culture medium for 1 h at 37°C. The cells were then seeded into the Elispot plate and cultured for 18 h at 37°C and 5% CO2. For each subject, triplicate wells were exposed to different stimulators. After culture, the plates were washed and incubated first with 0.2 µg/ml biotinylated detector Ab (Mabtech), then with 1:5000 streptavidin-alkaline phosphatase (Mabtech), and finally with BCIP/NBT (Sigma-Aldrich). The Elispot plates were analyzed with an automated imaging system and appropriate computer software (KS ELISPOT automated image analysis system, Zeiss, Jena, Germany). The frequency of cytokine-producing cells was expressed as the difference between the mean number of spots after stimulation and the mean background for each experiment.

Tissue samples
Human tonsils were obtained after tonsillectomies. The spleen was freshly obtained from an organ donor, who had died of cerebral hemorrhage.

Immunohistochemistry
Tissue samples were frozen immediately in Tissue tek® (Sakura, Zoeterwoude, The Netherlands) on dry ice. The 8-µm sections were cut and mounted on positively charged slides (SuperFrost*/Plus®, Menzel, Braunschweig) and stored at –20°C until use. All immunostainings were made on frozen sections, since the tested anti-SLAM mAbs did not stain paraffin-embedded material. For all immunostainings tissue sections were thawed, dried at 37°C for 15 min, washed in phosphate-buffered saline (PBS) and fixed in acetone for 10 min. They were then sequentially incubated with 1.5% hydrogen peroxidase for 10 min, 5% normal donkey serum for 30 min and the following mAbs for 2 h at room temperature or overnight at 4°C: anti-CD14-biotin (mAb X-8, diluted 1:200, BMA Biomedicals, Augst, Switzerland), anti-SLAM (IPO-3, diluted 1:100, Kamiya Biomedical, Seattle, WA), and anti-CD68 (1:1000) (Dako, Hamburg, Germany). The tissue sections were thoroughly washed for 5 min intervals in PBS and then incubated for 30 min in biotinylated donkey anti-mouse IgG antibody (1:500) (Jackson ImmunoResearch Laboratories). They were washed again in PBS and incubated with peroxidase-conjugated streptavidin (Dako) for 30 min, followed by a final wash and diaminobenzidine (DAB) substrate (Dako) for 10 min. Some sections were lightly counterstained with hematoxylin.

To perform a double immunostaining for SLAM and CD68, tissue sections were treated as described above but blocked in 5% normal goat serum. In a first step, the sections were incubated for 2 h with the anti-SLAM mAb, and then with peroxidase conjugated goat anti-mouse antibody (1:100) (Dako). The substrate staining was developed in a DAB solution, to which nickel chloride had been added (Vector Labs, USA). After incubation of the slides in this solution for 10 min, gray/black staining developed. After several 5 min washing intervals in distilled water and PBS, the sections were blocked with normal mouse serum (1:10) for 20 min (Dako), then washed in PBS and incubated with a peroxidase-conjugated CD68 antibody for 2 h at room temperature. Sections were then developed in normal DAB that results in a red/brown staining.

To perform a double immunofluorescence for SLAM and CD14, acetone fixed sections were washed in PBS, blocked with 5% normal goat serum and then incubated with the anti-SLAM mAb as described above. The SLAM-labeled cells were detected by using a Cy3-conjugated goat anti-mouse antibody (1:100). The sections were washed in PBS several times in the dark. In a second step after they had been blocked with normal mouse serum, sections were incubated with the biotinylated mouse anti-human CD14 antibody followed by several PBS washings and the labeling with Cy2-conjugated streptavidin (1:100) (Jackson ImmunoResearch Laboratories).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Quantitative differences in the induction of cytokine production by TLR-ligands and inflammatory cytokines
We analyzed quantitatively cytokine production of monocytes in response to different pathogen-derived products, each of which has been shown to be the ligand for a specific TLR: these included PGN and LTA as TLR-2 ligands, LPS and flagellin as TLR-4 and TLR-5 ligands, respectively. We compared the strength of these stimulations with inflammatory cytokines. Different concentrations of TLR-ligands (0.1–1 to 10 µg/ml) as well as IFN-{gamma} (500–2000 U/ml) and GM-CSF (10–50 ng/ml) were tested. Two different read out systems, the intracellular FACS staining and the Elispot were applied. Figure 1(A) shows an example of intracellular TNF-{alpha} FACS staining of 4 h-stimulated PBMC. Upon TLR-2, -4 or -5 ligation, 20–70% of monocytes started to produce TNF-{alpha} with the specific ligand, while only 2–5% responded after stimulation with high concentrations of IFN-{gamma} and GM-CSF (Fig. 1A). These frequencies were confirmed also by TNF-{alpha} Elispot assay on positively selected monocytes (Fig. 1B). About 1000 CD14+ monocytes were seeded in the Elispot plate and stimulated overnight. The strong response induced by TLR-ligands resulted in saturation in the number of spots/well. In this experiment, cells were not further diluted in order to detect the much weaker effects mediated by IFN-{gamma} and GM-CSF (only 1–2% of monocytes started producing TNF-{alpha}). Higher doses of the inflammatory cytokines did not provoke stronger stimulations (Fig. 1B).



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Fig. 1. Cytokine induction via TLR ligands and inflammatory cytokines. (A) TNF-{alpha} intracellular FACS staining on 4 h-stimulated PBMC. In the upper right quadrants the percentage of cells producing TNF-{alpha} is given. TLR-2, -4 or -5 ligation triggered TNF-{alpha} production in 20–70% of monocytes, while stimulation with IFN-{gamma} and GM-CSF activated only 2–5% of monocytes to produce TNF-{alpha}. (B) TNF-{alpha} Elispot assay of positively selected monocytes. Only a minority of CD14 positive cells produced this cytokine upon stimulation with inflammatory cytokines. The TNF-{alpha} production of monocytes in response to TLR-ligands was too strong to be counted by Elispot; to quantify the monocytic response to the TLR-ligands the TNF-{alpha} FACS is superior (A). (C) IL-10 Elispot assay of positively selected monocytes. <2% of monocytes produced IL-10 after TLR-2, -4 or -5 engagement. IFN-{gamma} and GM-CSF did not induce any IL-10 production.

 
Furthermore, we checked the production of the immunosuppressive cytokine IL-10 by Elispot assay on positively selected monocytes, and we found that LPS, PGN, LTA and flagellin induced release of IL-10, while inflammatory cytokines did not (Fig. 1C); however, in comparison with TNF-{alpha} secretion, only a small minority of monocytes (<2%) produced IL-10 upon TLR engagement.

Differential regulation of surface markers on monocytes in response to inflammatory cytokines and TLR ligands
We studied the regulation of expression of several monocyte surface markers in different stimulation conditions. Both inflammatory mediators and TLR ligands induced the expression of CD25, CD69, CD80 and CD83 with a variable intensity depending on the stimulus (Table 1 and Fig. 2). Since CD83 is commonly considered a marker for mature myeloid dendritic cells (DC) and not a monocyte activation marker, we elaborated our finding by using a different CD83-specific mAb in combination with different anti-CD14 mAb (Fig. 3). Kinetic analysis showed that CD83 expression on activated monocytes was very transient, appearing one day after activation and completely disappearing on day 2 (data not shown).


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Table 1. SLAM is distinct from other monocyte activation markers
 


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Fig. 2. Differential regulation of activation marker and costimulatory molecule expression by TLR ligands and inflammatory cytokines. PBMC were stimulated with IFN-{gamma} (as representative inflammatory cytokine), with LPS (as representative TLR ligand), or left untreated. After 18 h the cells were stained with the indicated mAbs. The percentage of positive cells is indicated, for CD86 staining the mean fluorescence intensity (MFI) is given. Both IFN-{gamma} and LPS induced CD25, CD69, and CD80 on monocyte surface, although with different intensities. IFN-{gamma} upregulated CD38, CD86, and ICOS-L, while LPS had the opposite effect. SLAM was the only marker induced by LPS and not by IFN-{gamma}.

 


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Fig. 3. CD83 is a monocyte activation marker. PBMC were stimulated for 18 h with IFN-{gamma}, LPS, or left untreated. After culture, cells were double-stained with mAbs specific for CD83 and CD14. IFN-{gamma}, and to a lesser degree LPS, induced CD83 on CD14-positive monocytes.

 
CD38, CD86 and ICOS-L were already present on the majority of monocytes and were downregulated by TLR ligands. The inflammatory cytokines induced a differential response: CD38 was upregulated by IFN-{gamma} and IFN-ß, but downregulated by GM-CSF and TNF-{alpha}; all these cytokines induced CD86 expression on monocytes, while only IFN-{gamma}, GM-CSF and IL-1 induced higher expression of ICOS-L.

SLAM expression is preferentially induced after engagement of TLRs
SLAM was a costimulatory molecule preferentially triggered by LPS and not by IFN-{gamma} (Fig. 2). In addition to LPS, the ligands of TLR-2 (LTA and PGN) and TLR-5 (flagellin) induced SLAM expression to a similar extent as LPS (Table 1). By contrast, ligands for TLR-3 [poly (I:C)] and TLR-9 (CpG ODN), did not induce SLAM on monocytes (data not shown). This is in line with the reported selective expression of TLR-2, -4 and -5 on monocytes (16).

To exclude the possibility that the stimulatory effect of the PGN, LTA and flagellin preparations was due to LPS contamination, we determined the minimal amount of TLR ligand necessary to induce SLAM and analyzed SLAM induction by these compounds in the presence of the LPS inhibitor polymixin B (10 µg/ml). SLAM induction started with 0.5 µg/ml PGN, LTA, flagellin and 100 pg/ml LPS, a concentration far above the endotoxin level present in the applied LTA, PGN and flagellin preparations; SLAM was induced by these three molecules even after blockade of LPS activity (data not shown).

We then tested the SLAM induction on monocytes in response to a greater panel of activators. SLAM was neither induced by such cytokines as IFN-{gamma}, IL-1ß, TNF-{alpha}, IFN-ß, GM-CSF (Table 1), nor by the interleukins IL-2, IL-4, IL-5, IL-7, IL-12, IL-17, nor by biologically active peptides of C3a and C5a, which function as proinflammatory anaphylatoxins, nor by heat shock proteins (bovine {alpha}B-crystallin, M. bovis BCG hsp65, human hsp60 and hsp70), nor by stimulation with an anti-CD40 mAb or with crosslinked recombinant human CD40L (Table 1 and data not shown). All these compounds that were unable to induce SLAM on monocytes were effective either in triggering cytokine production or in modulating the expression of other surface markers in PBMC cultures (data not shown). It seems therefore that SLAM expression is triggered preferentially upon TLR-2, -4 and -5 engagements.

We observed however that a polyclonal T cell activation of PBMC with solid-phase bound anti-CD3 induced SLAM on monocytes separated by a transwell from the activated PBMC. Further experiments showed that a combination of CD40-L + IL-1/IFN-{gamma} induced SLAM on monocytes in PBMC cultures, albeit to a lower degree than after LPS stimulation (Fig. 4, left panels). When CD14 positive cells were isolated and stimulated, they presented SLAM on the surface only upon exposure to LPS and at a lower extent to CD40-L + IL-1 (Fig. 4, right panels).



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Fig. 4. Differential SLAM induction on monocytes in PBMC or in purified CD14 positive cell culture. PBMC or purified CD14 positive cells were stimulated for 18 h as indicated or left untreated. The CD40-mediated activation was achieved with recombinant CD40L-FLAG crosslinked with anti-FLAG mAb. After culture, cells were stained with PE-labeled anti-human SLAM or with the corresponding isotype control. The percentage of positive cells is indicated. Single stimulation with IL-1ß, IFN-{gamma} (Table 1), or CD40L (this figure) did not induce SLAM on monocytes; however a combination of these activators did it in PBMC culture (left panels). On the other hand, a direct effect on monocytes was mediated only by LPS and at a lower extent by crosslinked CD40L+IL-1ß (right panels).

 
The expression of activation-induced SLAM on monocytes was transient. The majority (>50%) of monocytes were positive on day 1 during LPS stimulation, but already negative at day 2 (<10%) (Fig. 5). Stimulation with PGN, LTA or flagellin also induced a transient SLAM expression on monocytes with kinetics similar to LPS-induced SLAM expression (data not shown).



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Fig. 5. Transient expression of SLAM on blood-derived monocytes. PBMC were stimulated with LPS for 18 h (upper right panel) or 42 h (lower right panel). The left panels show the unstimulated monocytes. After culture, cells were stained with PE-labeled anti-human SLAM or with the corresponding isotype control. The large majority of monocytes was positive on day 1 after LPS stimulation, but already negative on day 2.

 
TLR-induced SLAM on monocytes requires activation of p38 MAPK and is independent of NF-{kappa}B activation
It is well established that cytokine production by monocytes via LPS requires the activation of the three MAPK signaling pathways. To determine whether these pathways were also necessary for the induction of SLAM expression on monocytes, PBMC were stimulated with LPS alone, or in the presence of herbimycin A (inhibitor for all tyrosine kinases), SB 203580 (specific inhibitor for p38 MAPK), PD 98059, U0126 (specific inhibitors of MEK1/2 activating ERK kinases), or dexamethasone (inhibitor of JNK kinases). All three MAPK pathways were found to be involved in LPS, LTA, PGN and flagellin-mediated TNF-{alpha} (data not shown) and IL-10 production (shown for LPS in Fig. 6A). On the other side, only herbimycin A and SB 203580 were able to block SLAM induction (Fig. 6B), indicating that among the MAPK pathways only p38 signaling is necessary for SLAM expression on the monocyte surface after TLR-4 triggering. TLR-2 engagement by LTA and PGN, and TLR-5 by flagellin also induced SLAM in a p38-dependent manner (data not shown).



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Fig. 6. IL-10 production is mediated via the three MAPK pathways and NF-{kappa}B, while SLAM induction is mediated via the MAPK p38 and is independent of NF-{kappa}B. IL-10 Elispot assay on PBMC. Cells were seeded in the Elispot wells and cultured overnight alone, with LPS or with LPS + specific inhibitors. IL-10 production was inhibited by herbimycin A (HA; inhibitor of tyrosine kinases), SB 203580 (specific inhibitor of p38 MAPK), dexamethasone (Dex; inhibitor of JNK kinases), PD 98059 and UO 126 (inhibitors of ERK kinases). PBMC were stimulated for 18 h with 300 ng/ml LPS alone or in the presence of specific inhibitors. After culture, cells were stained with PE-labeled anti-SLAM or with the corresponding isotype control. The percentage of cells expressing SLAM is given in the upper right quadrants. SLAM induction was inhibited by herbimycin A (HA) and SB 203580 but not by dexamethasone (Dex), PD 98059 and PDTC. Another inhibitor of ERK kinases, U0126, was also tested and did not inhibit SLAM induction (data not shown). IL-10 Elispot assay on PBMC. Cells were seeded in the Elispot wells and cultured overnight alone, with TLR-ligands or with TLR-ligands + PDTC (specific NF-{kappa}B inhibitor). PDTC blocked TLR-ligands-triggered IL-10 secretion by monocytes.

 
To address the role of NF-{kappa}B in SLAM induction on monocytes, we applied different concentrations (1–100 µM) of PDTC, a specific NF-{kappa}B inhibitor (17). At a concentration of 1 µM PDTC, the LPS-induced cytokine production was strongly reduced (data not shown). At a concentration of 10 µM PDTC, no effect on the SLAM expression induced by LPS (Fig. 6C, lower panel) or PGN, LTA, flagellin (data not shown) was detectable. However, PDTC could almost completely inhibit the TNF-{alpha} (data not shown) and IL-10 induction by LPS, LTA, PGN or flagellin (Fig. 6C), demonstrating that SLAM induction was an NF-{kappa}B-independent mechanism.

SLAM-bearing monocytes are absent in blood, but present in tonsils and spleen
Monocytes in blood did not express SLAM without further stimulation (Figs 2 and 46). In tonsils, SLAM+ monocytes were detected by immunohistochemistry and by FACS. Figure 7 (upper left panel) shows one (out of three) representative FACS analysis of tonsil-derived cells. Although present in small amount in tonsils, almost all CD14+ monocytes stained positive for SLAM. The immunohistochemical stainings showed that in the tonsils, SLAM+ cells were scattered in the T and B cell areas and concentrated in the mantel zone around the germinal centers (Fig. 7B). CD14 staining was observed in most of the germinal centers and in the paracortex (Fig. 7A). Some of the CD14+ cells in the paracortex also stained strongly for SLAM (Fig. 7D), while CD14+ cells from the germinal center showed a weaker SLAM staining (Fig. 7E). CD68+ macrophages were localized in the germinal centers and in the paracortex (Fig. 7C). Double staining with CD14 showed that CD68+ macrophages were distinct from CD14+ monocytes (Fig. 7F). Double staining with SLAM could localize CD68+ cells expressing SLAM in the germinal centers (Fig. 7G).



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Fig. 7. SLAM positive monocytes in tonsils and spleen. Tonsils and spleen were mechanically disrupted, and single-cell suspensions of tonsils were subjected to the Lymphoprep gradient. Subsequently FACS analysis was done as described in Methods. The numbers in the upper quadrants in each panel give the distribution of SLAM+ and SLAM– cells. Frozen sections of tonsils were stained with mAb to CD14 (A), SLAM (B), and CD68 (C). CD14+ and CD68+ cells could be seen in the germinal center and in the T cell area. SLAM+ cells were detected throughout the tonsil, but there was a particularly strong staining in the mantel layer of the germinal centers. CD14-expressing cells were labeled green (D–F). SLAM (D and E) or CD68 (F) expressing cells were labeled red. CD14+ cells, which strongly express SLAM (yellow in D), were found outside of the germinal center in the T cell area. Panels (E) and (F) are from the same region of the germinal center shown in (A). CD14+ cells in the germinal centers exhibited a lower intensity of SLAM expression, which resulted in a yellowish color (E). Double staining of CD14 and CD68 revealed that CD14 and CD68 were expressed by different cells, since only red and green, but no yellowish cells appeared (F). (G) SLAM-expressing cells were stained blue and CD68-expressing cells brown. Many small blue cells expressing only SLAM are visible. Two CD68+ cells co-expressing SLAM (arrow) and one CD68+ cell not expressing SLAM (arrowhead) are shown. Original magnification was 100x (A–C), 400x (D–G) or 1000x (E).

 
FACS analyses of human spleen revealed that about half of the monocytes in this lymphoid organ expressed SLAM (Fig. 7, uper right panel).


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study we have analyzed the response of human monocytes to bacterial stimuli and report the following novel findings: i) TLR-2, -4 and -5 engagement activated ~50% of the monocytes to produce TNF-{alpha} (compared to only 5% of monocytes after induction with IFN-{gamma} or GM-CSF) and <2% to secrete IL-10; ii) while both TLR-ligands and inflammatory cytokines induced CD25, CD69, CD80 and, surprisingly, CD83 on monocytes, only TLR ligands tended to downregulate CD38, CD86 and ICOS-L; iii) SLAM is distinct from other monocyte activation markers, since it is induced ex novo preferentially via TLR-2, -4 and -5; iv) TLR-mediated SLAM induction requires the MAPK p38, not ERK or JNK, and is independent of NF-{kappa}B; v) SLAM positive monocytes, which are absent in blood, were present in tonsils and spleen.

Modulation of cytokine production, activation markers and costimulatory molecules on human monocytes
The usage of the same experimental asset for several activators allowed us to point out fundamental differences about activation pathways of human monocytes. We looked at the changes both in the expression of membrane molecules and at the level of cytokine secretion. Information about monocyte reactivity is very fragmented and often obtained by the analysis of cell lines and not of human fresh cells. It is known, for example, that TLR ligands induce production of cytokines such as TNF-{alpha}, but the strength of these stimuli in comparison with other physiological activators was never quantified. In fact we found that TLR-ligands are more potent inducers of TNF-{alpha} production in comparison with inflammatory cytokines, as IFN-{gamma} and GM-CSF. Secondly, Elispot assays revealed that a small minority (<2%) of monocytes started to produce the immunosuppressive cytokine IL-10 upon TLR ligation, while the rate of TNF-{alpha} producing monocytes was at least 10-fold higher in response to the same stimulus. This is in line with the fact that after TLR engagement activating effects far exceed inhibitory effects. IL-10 production may serve to limit a bacteria-mediated inflammatory immune response, since the reduced ability of monocytes to present antigen during endotoxin tolerance is mediated by IL-10 (18).

Very little is known about the regulation of expression of surface markers on freshly-isolated human monocytes. We noted that inflammatory cytokines like IFN-{gamma} on one side and some TLR-ligands on the other side have similar, but also opposite properties. Only a minority of circulating monocytes expressed IL-2R (CD25), CD69 and B7.1 (CD80), and we found that these markers could be induced after a short in vitro treatment with either monocyte-activating cytokines or with TLR-ligands. We noted that CD83, commonly used as a specific marker for mature DC (19) is also a monocyte activation marker. Mature monocyte-derived DC lose the expression of CD14, but start to express CD83 and SLAM (7). The usage of two different anti-CD83 mAbs and double staining with CD14 revealed that CD83 was induced on CD14+ monocytes. This activation-induced expression of CD83 was very ephemeral, appearing on day 1 and disappearing on day 2. Our findings agree with those of a recent microarray study that found SLAM and CD83 on monocyte-derived macrophages after stimulation with bacterial components (20). The recent report that the human CD83 promoter contained an NF-{kappa}B element and is inducible by TNF-{alpha} (21), is also in line with our results.

CD38, B7.2 (CD86) and ICOS-L were constitutively expressed in vivo by a large proportion of monocytes and were downregulated by TLR-ligands. The finding that IFN-{gamma}, GM-CSF and IL-1 upregulated ICOS-L, while IFN-ß and TNF-{alpha} had no effect, confirms and extends previous observations (22). Since ICOS-L-mediated costimulation plays a strong role in IL-10 production by T cells (15), the TLR-mediated downregulation of ICOS-L on monocytes might be part of the strong proinflammatory effect mediated by TLRs.

SLAM was induced by TLRs on monocytes. Since SLAM is a self ligand (6,14), SLAM-expressing monocytes may therefore costimulate SLAM-expressing T and B cells. On T cells, SLAM ligation strongly promotes Th1 development (5). This contributes to a Th1-promoting microenvironment after TLR engagement of the monocytes.

Regulation of SLAM-induction on human monocytes
The present study revealed that SLAM is distinct from other monocyte activation markers (Table 1). Ligands for TLR-2, -4 and -5, but not for TLR-3 and -9 [poly (I:C) and CpG ODN] induced SLAM on monocytes. This is in line with the reported selective expression of TLR-2, -4 and -5 on monocytes (16). Other kinds of stimuli for monocytes were tested (inflammatory and anti-inflammatory cytokines, complement factor-derived peptides, heat shock proteins and CD40 engagement), but none was able to mediate the same effect. IL-1 or CD40-ligation, which enhance SLAM expression on DC (7,8), did not induce SLAM on monocytes, if not in combination and then at a lower extent in comparison to LPS. Indeed, some responses of monocytes and immature DC indicated differences in responses to the same inflammatory stimulus: whereas LPS upregulates CD86 on immature DC, it downregulates it on monocytes (23). The induction of SLAM on monocytes via TLR ligands was transient. This contrasts to the kinetics of SLAM on T cells (24), B cells (6), and DC (8), which continue to express SLAM for a longer time after activation.

Some recent reports suggest the existence of endogenous ligands for TLRs, such as HSP60 (25) and HSP70 (26). Our observation that LPS-free HSP60 and HSP70 did not induce SLAM on monocytes and failed to stimulate cytokine production (C. Farina, unpublished results) sheds new light on the debate about HSPs as monocyte activators and as possible ligands for TLRs (27,28). In addition to HSPs, fibronectin (29), heparan sulfate proteoglycan (30), oligosaccharides of hyaluronan (31) and necrotic (not apoptotic) material (32) were reported to be endogenous ligands for TLRs. These reports agree with the view that ‘danger’ signals alert the immune system (33).

Differential modes of monocyte intervention
We can therefore imagine two modes of immune intervention by monocytes: on one side, upon bacterial trigger they downregulate CD38, CD86, ICOS-L, express transiently SLAM that ‘force’ activated T cells to a TH1 phenotype. Monocytes produce huge amounts of inflammatory cytokines, but some also start secreting anti-inflammatory mediators. On the other side, an inflammatory environment stimulates monocytes to upregulate CD38, CD86 and ICOS-L, but only a few can produce inflammatory cytokines. The first mode would allow a rapid, strong and direct attack of infectious agents, while the second would better support the intervention of cells of the adaptive arm.

Different signaling requirements for SLAM induction and cytokine production
To determine the signal transduction elements necessary for SLAM induction, we dissected the three MAPK signaling pathways by specific inhibitors. Cytokine production and SLAM induction were mediated via different signaling pathways. While production of TNF-{alpha} and IL-10 upon TLR-engagement involved all three MAPK pathways (34), SLAM induction required only the activation of p38 MAPK. Very little is known about the signaling machinery necessary for the expression of costimulatory molecules. A recent study demonstrated that LPS-mediated CD86 downregulation depended on p38 and JNK, but not ERK (23). The activation of NF-{kappa}B is considered a hallmark of TLR-mediated effects (35). We found that SLAM induction was not dependent on NF-{kappa}B activity, whereas cytokine production was. This demonstrates that ligands of TLR-2, -4 and -5 could mediate effects independently of NF-{kappa}B activity on human monocytes. In agreement with this observation, the NF-{kappa}B inhibitor PDTC in mouse peritoneal macrophages did not affect the nuclear translocation of interferon-regulatory factor-3 upon stimulation with Lipid A (36). LPS induces diverse signaling pathways that involve distinct mediators such as Tollip, TIRAP, PKR and MyD88 (37); their role in SLAM induction remains to be identified. As monocytes and macrophages do not contain internal stores of SLAM molecules (38), other studies are needed to identify the transcription factors in the downstream signaling leading to SLAM induction. Since this study showed the selective dependence of SLAM induction on the MAPK p38, likely candidate transcription factors are ATF2, Elk-1 and MEF-2C (34).

In vivo distribution of SLAM positive monocytes and macrophages
To relate the in vitro findings to SLAM expression in vivo, we looked for SLAM+ monocytes in vivo in secondary lymphoid organs. The vast majority of tonsillar monocytes and about half of the splenic monocytes were SLAM+. In tonsils, SLAM+ monocytes were localized not only in T cell areas, but also within germinal centers suggesting they play a role in B cell activation. SLAM+ macrophages (CD68+) were also found in germinal centers. The expression of SLAM on tonsillar monocytes and macrophages can be readily attributed to TLR-mediated activation due to the presence of normal bacterial flora. The SLAM positive monocytes in the spleen could be explained by its role in scavenging bacteria. In fact, splenectomized individuals are prone to overwhelming infections with encapsulated bacteria, and splenectomy of mice increases susceptibility to streptococcal infections. The clearing of endogenous necrotic material might in addition induce SLAM on splenic monocytes, since necrotic cells can activate TLRs (32).

This paper reports that SLAM expression on monocytes is preferentially induced via ligands of TLR-2, -4 and -5, rather than by inflammatory cytokines. This leads to the hypothesis that SLAM expression on monocytes in vivo is linked to bacterial infection, but not necessarily to inflammation. This hypothesis was tested and indeed SLAM was abundantly expressed in inflammatory settings with bacterial exposure, but not in inflammatory settings of autoimmune pathogenesis (manuscript in preparation).

TLR engagement also modulates the expression of chemokine receptors that are involved in monocyte recruitment, e.g. CCR2 and CXCR3 (C. Farina, unpublished results). Thus, in addition to the local microenvironment, monocyte migration modified by TLR engagement determines the localization of SLAM+ monocytes.

In summary, we have identified an activation marker on monocytes, SLAM that is distinct from other monocyte activation markers, since it was preferentially induced by TLR-2, -4 and -5 ligands, but not by single inflammatory cytokines. SLAM+ monocytes are absent in blood, but detected in secondary lymphoid organs. Monocytes expressing SLAM might therefore serve as a sign of innate immune activation. SLAM induction on monocytes was mediated by MAPK p38, and—unusual for TLR-mediated effects—independently of NF-{kappa}B.


    Acknowledgements
 
We thank Mrs D. Zech, Drs M. Heiss and H. Hartl (University of Munich) for their help, and J. Benson, A. Flügel, A. Holz for reading the manuscript. This work was supported by the DFG (SFB 571). The Institute for Clinical Neuroimmunology is supported by the Hermann and Lilly Schilling Foundation.


    Abbreviations
 
DC—dendritic cell

HSP—heat shock protein

LTA—lipoteichoic acid

MAPK—mitogen-activated protein kinase

PGN—peptidoglycan

SLAM—signaling lymphocytic activation molecule

TLR—Toll-like receptor


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Medzhitov, R. and Janeway, C. A. Jr 2002. Decoding the patterns of self and nonself by the innate immune system. Science 296:298.[Abstract/Free Full Text]
  2. Underhill, D. M. and Ozinsky, A. 2002. Toll-like receptors: key mediators of microbe detection. Curr. Opin. Immunol. 14:103.[CrossRef][ISI][Medline]
  3. Watts, T. H. and DeBenedette, M. A. 1999. T cell co-stimulatory molecules other than CD28. Curr. Opin. Immunol. 11:286.[CrossRef][ISI][Medline]
  4. Veillette, A. and Latour, S. 2003. The SLAM family of immune-cell receptors. Curr. Opin. Immunol. 15:277.[CrossRef][ISI][Medline]
  5. Cocks, B. G., Chang, C. C., Carballido, J. M., Yssel, H., de Vries, J. E. and Aversa, G. 1995. A novel receptor involved in T-cell activation. Nature 376:260.[CrossRef][ISI][Medline]
  6. Punnonen, J., Cocks, B. G., Carballido, J. M., Bennett, B., Peterson, D., Aversa, G. and de Vries, J. E. 1997. Soluble and membrane-bound forms of signaling lymphocytic activation molecule (SLAM) induce proliferation and Ig synthesis by activated human B lymphocytes. J. Exp. Med. 185:993.[Abstract/Free Full Text]
  7. Kruse, M., Meinl, E., Henning, G., Kuhnt, C., Berchtold, S., Berger, T., Schuler, G. and Steinkasserer, A. 2001. Signaling lymphocytic activation molecule is expressed on mature CD83+ dendritic cells and is up-regulated by IL-1ß. J. Immunol. 167:1989.[Abstract/Free Full Text]
  8. Bleharski, J. R., Niazi, K. R., Sieling, P. A., Cheng, G. and Modlin, R. L. 2001. Signaling lymphocytic activation molecule is expressed on CD40 ligand-activated dendritic cells and directly augments production of inflammatory cytokines. J. Immunol. 167:3174.[Abstract/Free Full Text]
  9. Murabayashi, N., Kurita-Taniguchi, M., Ayata, M., Matsumoto, M., Ogura, H. and Seya, T. 2002. Susceptibility of human dendritic cells (DCs) to measles virus (MV) depends on their activation stages in conjunction with the level of CDw150: role of Toll stimulators in DC maturation and MV amplification. Microbes Infect. 4:785.[CrossRef][ISI][Medline]
  10. Minagawa, H., Tanaka, K., Ono, N., Tatsuo, H. and Yanagi, Y. 2001. Induction of the measles virus receptor SLAM (CD150) on monocytes. J. Gen. Virol. 82:2913–2917.[Abstract/Free Full Text]
  11. Howie, D., Okamoto, S., Rietdijk, S., Clarke, K., Wang, N., Gullo, C., Bruggeman, J. P., Manning, S., Coyle, A. J., Greenfield, E., Kuchroo, V. and Terhorst, C. 2002. The role of SAP in murine CD150 (SLAM)-mediated T-cell proliferation and interferon gamma production. Blood 100:2899.[Abstract/Free Full Text]
  12. Henning, G., Kraft, M. S., Derfuss, T., Pirzer, R., Saint-Basile, G., Aversa, G., Fleckenstein, B. and Meinl, E. 2001. Signaling lymphocytic activation molecule (SLAM) regulates T cellular cytotoxicity. Eur. J. Immunol. 31:2741.[CrossRef][ISI][Medline]
  13. Tatsuo, H., Ono, N., Tanaka, K. and Yanagi, Y. 2000. SLAM (CDw150) is a cellular receptor for measles virus. Nature 406:893.[CrossRef][ISI][Medline]
  14. Mavaddat, N., Mason, D. W., Atkinson, P. D., Evans, E. J., Gilbert, R. J., Stuart, D. I., Fennelly, J. A., Barclay, A. N., Davis, S. J. and Brown, M. H. 2000. Signaling lymphocytic activation molecule (SLAM, CDw150) is homophilic but self-associates with very low affinity. J. Biol. Chem. 275:28100.[Abstract/Free Full Text]
  15. Witsch, E. J., Peiser, M., Hutloff, A., Buchner, K., Dorner, B. G., Jonuleit, H., Mages, H. W. and Kroczek, R. A. 2002. ICOS and CD28 reversely regulate IL-10 on re-activation of human effector T cells with mature dendritic cells. Eur. J. Immunol. 32:2680.[CrossRef][ISI][Medline]
  16. Hornung, V., Rothenfusser, S., Britsch, S., Krug, A., Jahrsdorfer, B., Giese, T., Endres, S. and Hartmann, G. 2002. Quantitative expression of toll-like receptor 1–10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J. Immunol. 168:4531.[Abstract/Free Full Text]
  17. Schreck, R., Meier, B., Mannel, D. N., Droge, W. and Baeuerle, P. A. 1992. Dithiocarbamates as potent inhibitors of nuclear factor kappa B activation in intact cells. J. Exp. Med. 175:1181.[Abstract]
  18. Wolk, K., Docke, W. D., von Baehr, V., Volk, H. D. and Sabat, R. 2000. Impaired antigen presentation by human monocytes during endotoxin tolerance. Blood 96:218.[Abstract/Free Full Text]
  19. Zhou, L. J. and Tedder, T. F. 1996. CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells. Proc. Natl Acad. Sci. USA 93:2588.[Abstract/Free Full Text]
  20. Nau, G. J., Richmond, J. F., Schlesinger, A., Jennings, E. G., Lander, E. S. and Young, R. A. 2002. Human macrophage activation programs induced by bacterial pathogens. Proc. Natl Acad. Sci. USA 99:1503.[Abstract/Free Full Text]
  21. Berchtold, S., Muhl-Zurbes, P., Maczek, E., Golka, A., Schuler, G. and Steinkasserer, A. 2002. Cloning and characterization of the promoter region of the human CD83 gene. Immunobiology 205:231.[ISI][Medline]
  22. Aicher, A., Hayden-Ledbetter, M., Brady, W. A., Pezzutto, A., Richter, G., Magaletti, D., Buckwalter, S., Ledbetter, J. A. and Clark, E. A. 2000. Characterization of human inducible costimulator ligand expression and function. J. Immunol. 164:4689.[Abstract/Free Full Text]
  23. Lim, W., Ma, W., Gee, K., Aucoin, S., Nandan, D., Diaz-Mitoma, F., Kozlowski, M. and Kumar, A. 2002. Distinct role of p38 and c-Jun N-terminal kinases in IL-10-dependent and IL-10-independent regulation of the costimulatory molecule B7.2 in lipopolysaccharide-stimulated human monocytic cells. J. Immunol. 168:1759.[Abstract/Free Full Text]
  24. Aversa, G., Chang, C. C., Carballido, J. M., Cocks, B. G. and de Vries, J. E. 1997. Engagement of the signaling lymphocytic activation molecule (SLAM) on activated T cells results in IL-2-independent, cyclosporin A-sensitive T cell proliferation and IFN-gamma production. J. Immunol. 158:4036.[Abstract]
  25. Ohashi, K., Burkart, V., Flohe, S. and Kolb, H. 2000. Cutting edge: heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex. J. Immunol. 164:558.[Abstract/Free Full Text]
  26. Asea, A., Kraeft, S. K., Kurt-Jones, E. A., Stevenson, M. A., Chen, L. B., Finberg, R. W., Koo, G. C. and Calderwood, S. K. 2000. HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine. Nat. Med. 6:435.[CrossRef][ISI][Medline]
  27. Wallin, R. P., Lundqvist, A., More, S. H., von Bonin, A., Kiessling, R. and Ljunggren, H. G. 2002. Heat-shock proteins as activators of the innate immune system. Trends Immunol. 23:130.[CrossRef][ISI][Medline]
  28. Bausinger, H., Lipsker, D. and Hanau, D. 2002. Heat-shock proteins as activators of the innate immune system. Trends Immunol. 23:342.[CrossRef][ISI][Medline]
  29. Smiley, S. T., King, J. A. and Hancock, W. W. 2001. Fibrinogen stimulates macrophage chemokine secretion through toll-like receptor 4. J. Immunol. 167:2887.[Abstract/Free Full Text]
  30. Johnson, G. B., Brunn, G. J., Kodaira, Y. and Platt, J. L. 2002. Receptor-mediated monitoring of tissue well-being via detection of soluble heparan sulfate by Toll-like receptor 4. J. Immunol. 168:5233.[Abstract/Free Full Text]
  31. Termeer, C., Benedix, F., Sleeman, J., Fieber, C., Voith, U., Ahrens, T., Miyake, K., Freudenberg, M., Galanos, C. and Simon, J. C. 2002. Oligosaccharides of Hyaluronan activate dendritic cells via toll-like receptor 4. J. Exp. Med. 195:99.[Abstract/Free Full Text]
  32. Li, M., Carpio, D. F., Zheng, Y., Bruzzo, P., Singh, V., Ouaaz, F., Medzhitov, R. M. and Beg, A. A. 2001. An essential role of the NF-kappa B/Toll-like receptor pathway in induction of inflammatory and tissue-repair gene expression by necrotic cells. J. Immunol. 166:7128.[Abstract/Free Full Text]
  33. Matzinger, P. 2002. The danger model: a renewed sense of self. Science 296:301–305.[Abstract/Free Full Text]
  34. Dong, C., Davis, R. J. and Flavell, R. A. 2002. MAP kinases in the immune response. Annu. Rev. Immunol. 20:55.[CrossRef][ISI][Medline]
  35. Aderem, A. and Ulevitch, R. J. 2000. Toll-like receptors in the induction of the innate immune response. Nature 406:782.[CrossRef][ISI][Medline]
  36. Kawai, T., Takeuchi, O., Fujita, T., Inoue, J., Muhlradt, P. F., Sato, S., Hoshino, K. and Akira, S. 2001. Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes. J. Immunol. 167:5887.[Abstract/Free Full Text]
  37. Henneke, P. and Golenbock, D. T. 2001. TIRAP: how Toll receptors fraternize. Nat. Immunol 2:828.[CrossRef][ISI][Medline]
  38. Kurita-Taniguchi, M., Hazeki, K., Murabayashi, N., Fukui, A., Tsuji, S., Matsumoto, M., Toyoshima, K. and Seya, T. 2002. Molecular assembly of CD46 with CD9, {alpha}3-ß1 integrin and protein tyrosine phosphatase SHP-1 in human macrophages through differentiation by GM-CSF. Mol. Immunol. 38:689.[CrossRef][ISI][Medline]