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The Journal of Immunology, 2003, 171: 3955-3962.
Copyright © 2003 by The American Association of Immunologists

Functional Effects of TNF-{alpha} on a Human Follicular Dendritic Cell Line: Persistent NF-{kappa}B Activation and Sensitization for Fas-Mediated Apoptosis 1

Sun-Mi Park*, Hae-Young Park{dagger} and Tae H. Lee2,*

* Department of Biology and Protein Network Research Center, Yonsei University, Seoul, Korea; and {dagger} Department of Biochemistry, School of Medicine, Ewha Woman’s University, Seoul, Korea


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Follicular dendritic cells (FDC) play crucial roles in germinal center (GC) formation and differentiation of GC B cells. FDC functions are influenced by cytokines produced in the GC. Among the GC cytokines, TNF is known to be essential for the formation and maintenance of the FDC network in the GC. We found that TNF is a mitogenic growth factor to an established FDC-like cell line, HK cells. Differing from most cell types which become desensitized to TNF action, HK cells exhibited persistent TNF signaling, as demonstrated by prolonged and biphasic NF-{kappa}B activation even after 3 days of TNF treatment. As a result, antiapoptotic genes including TNFR-associated factors 1 and 2, and cellular inhibitor of apoptosis proteins 1 and 2 were persistently induced by TNF, leading to the protection against TNF-mediated cell death. However, TNF pretreatment enhanced Fas-mediated apoptosis by up-regulating surface Fas expression in an NF-{kappa}B-dependent pathway. During the GC responses, proliferation followed by FDC death has not been documented. However, our in vitro results suggest that FDCs proliferate in response to TNF, and die by Fas-mediated apoptosis whose susceptibility is enhanced by TNF, representing a mode of action for TNF in the maintenance of FDC networks by regulating the survival or death of FDC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Germinal centers (GC) 3 in secondary lymphoid follicles provide a specialized microenvironment where Ag-specific B cells clonally expand and differentiate into memory cells or plasma cells (1, 2). Follicular dendritic cells (FDC) play essential roles in this sequential process of B cell differentiation, known as GC reaction. FDCs capture native Ag in the form of immune complexes and present them to GC B cells, thereby selecting cells with high affinity B cell receptors. Binding to FDC is a prerequisite for GC B cells to survive. Cells that do not succeed in binding to FDC die rapidly by apoptosis (3, 4, 5). Besides the Ag-specific interaction, FDCs also provide a set of adhesion molecules and cytokine signals to facilitate the appropriate interaction with GC B cells (6).

The function of FDC in supporting GC reaction is influenced by the action of cytokines present in GC microenvironments. Among the GC cytokines, TNF-{alpha} plays an important role in the induction and differentiation of FDC, as evidenced from the lack of FDC network in primary B cell follicles and failure to form GC after immunization in mice deficient in TNF or its receptor TNFR1 (7, 8, 9, 10). Although the functional importance of TNF signals in the induction and maintenance of FDC network is well-recognized, intracellular pathways and targets that are regulated by TNF in FDC activation have not been fully understood. The use of FDCs in culture is a valuable approach to study the function of TNF in FDC activation. Among the FDC-like lines, HK cells are relatively well-characterized. Their functional and phenotypic characteristics are shown to resemble many aspects of FDCs, delaying apoptosis and stimulating the growth and differentiation of GC B cells (11, 12, 13). Using this cell line, we have previously shown that TNF preferentially activates extracellular signal-regulated kinases 1 and 2 (ERK1/2) that are implicated in the cellular proliferation response (14). In the present study, we further examined the responses of HK cells to TNF. We showed that HK cells proliferated in response to TNF, and exhibited a sustained NF-{kappa}B activation due to persistent TNF signaling. The persistent TNF signaling resulted in a prolonged biphasic induction of genes including TNFR-associated factor (TRAF)1, TRAF2, cellular inhibitor of apoptosis protein (c-IAP)1 and c-IAP2, thus protecting HK cells from TNF-mediated apoptosis. We also found that TNF sensitizes HK cells to Fas-mediated apoptosis by up-regulating surface Fas expression in an NF-{kappa}B-dependent manner. During the GC reaction, FDC proliferates to form a FDC network and then apoptosis of FDC may occur in the resolution stage of GC responses. Our results suggest that FDC proliferation and death are regulated by TNF.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures

Cell culture media were purchased from Life Technologies (Rockville, MD). Two FDC-like HK cell lines established from tonsils of different donors were obtained from Dr. Y. S. Choi (Alton Ochsner Medical Foundation, New Orleans, LA) (11, 14) and Dr. J. Choe (Kang Won National University, Choonchun, Korea) (15), and were grown in RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. HK cells with passages 13–17 were used for various experiments. Human fetal lung fibroblast LF-1 cells (14) were grown in DMEM supplemented with 10% FCS.

Reagents

Recombinant soluble Fas ligand (sFasL) used in this study was prepared from the culture supernatant of the stable cell line, CHO-K1-sFasL cells, that was grown in serum-free medium (CHO-S-SFM II; Life Technologies). The biological activity of sFasL has been previously described (16). In experiments, cells were treated with the sFasL supernatant to the final concentration of 10%. Recombinant human TNF was obtained from Biotech Research Institute (LG Life Sciences, Seoul, Korea). N-acetylleucylleucylnorleucinal (ALLN), DMSO, and cyclocheximide (CHX) were purchased from Calbiochem (La Jolla, CA). Rabbit polyclonal I{kappa}B{alpha} (B-9) and Fas (C-29) Abs, and mAbs to NF-{kappa}B p65/RelA, caspase-3, and poly(ADP-ribose) polymerase (PARP) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The polyclonal anti-caspase-8 Ab was purchased from BD PharMingen (San Diego, CA). Polyclonal Ab against the serine-32-phosphorylated form of I{kappa}B{alpha} was from New England Biolabs (Beverly, MA). Adenovirus expressing flag-tagged I{kappa}B{alpha} super-repressor with alanine mutations at serine 32 and serine 36 or {beta}-galactosidase were kindly provided by Dr. C.-G. Yoo (College of Medicine, Seoul National University, Seoul, Korea) (17).

Preparation of nuclear extracts and EMSA

HK cells were treated with TNF at the concentration of 20 ng/ml for the indicated times. Nuclear extracts were prepared as described by Dignam et al. (18) and quantitated by the Bradford assay (Bio-Rad, Richmond, CA). The oligonucleotide probes for EMSA corresponded to the NF-{kappa}B binding sites in the c-IAP2 promoter (sense, 5'-ATGGAAATCCCCGA-3' and antisense, 5'-TCGGGGATTTCCAT-3') (19). Two oligonucleotides complementary to each other were annealed to generate a double-stranded probe. End-labeling was accomplished by treatment with T4 polynucleotide kinase (Roche Molecular Biochemicals, Indianapolis, IN) in the presence of [{gamma}-32P]ATP. One nanogram of the labeled probe was mixed with 5 µg of nuclear protein in a binding condition of 20 mM HEPES (pH 7.9), 60 mM KCl, 1 mM EDTA, 0.5 mM DTT, 10% glycerol, and 2 µg of poly(dI · dC). The reaction mixture was electrophoresed in a 6% nondenaturing polyacrylamide gel. The gel was vacuum-dried and subjected to autoradiography.

NF-{kappa}B localization study

HK cells were grown on glass coverslips precoated with 0.1% gelatin in a 24-well plate. After treatment with 20 ng/ml TNF for the indicated times, cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min, and permeabilized with PBS containing 0.1% Triton X-100 for 20 min. Subsequently, the permeabilized cells were blocked in TBS/Tween 20 supplemented with 5% skim milk for 1 h and then incubated with anti-p65/RelA Ab for 1 h at room temperature. Cells were then washed and incubated with a rhodamine-conjugated goat anti-mouse IgG secondary Ab for 1 h at room temperature. Cells were next washed with PBS containing 0.1% Triton X-100 and with PBS, and examined by fluorescence microscopy.

Western blot analysis

After stimulation, cells were washed with cold PBS, scraped, and resuspended in lysis buffer containing 1% Nonidet P-40, 50 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM EDTA, 1 mM pyrophosphate, 10 mM sodium orthovanadate, 3 mM benzamidine, 1 mM PMSF, and 100 mM sodium fluoride. Cell lysates were centrifuged at 15,000 rpm for 10 min at 4°C. The supernatants were electrophoresed through 10% SDS-PAGE. Proteins were transferred to nitrocellucose membrane (Bio-Rad). The blots were blocked in TBS/Tween 20 supplemented with 5% skim milk for 1 h, incubated with various primary Abs for 1 h and then with 1/5000 diluted secondary Abs of HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Santa Cruz Biotechnology) for 1 h at room temperature. The blots were treated with ECL reagents (Amersham Pharmacia Biotech, Piscataway, NJ) and detected by autoradiography.

RNase protection assay (RPA) and Northern blot analysis

Confluent cultures of HK cells in 100-mm dishes were stimulated with 20 ng/ml TNF for the indicated times. Total RNA was extracted using Tri-reagent (Molecular Research Center, Cincinnati, OH) and 10 µg of RNA were analyzed using the Riboquant multiprobe RNase protection assay kit (BD PharMingen) according to the manufacturer’s instructions. In brief, the human APO-5 multiprobe set was labeled with [{alpha}-32P]UTP. Sample RNA was hybridized overnight with the 32P-labeled probes and subjected to RNase digestion. The protected probes were resolved on a 6% denaturing polyacrylamide gel. Northern blot analysis for Fas mRNA was performed with 20 µg of total RNA samples prepared after treating HK cells as indicated. The radiolabeled DNA probe was made with the BglII fragment from human Fas cDNA (~650 bp) by using a random prime labeling kit (Amersham Pharmacia Biotech).

Measurement of apoptosis (nuclear staining with Hoechst 33258)

HK cells plated in 12-well plates were untreated or pretreated with 20 ng/ml TNF for 24 h before the sFasL stimuli. Cells given the death-inducing stimuli were then fixed with 4% paraformaldehyde for 20 min at room temperature and stained with 50 ng/ml Hoechst 33258 (Sigma-Aldrich, St. Louis, MO). For quantitation of apoptosis (presented as average ± SEM), cells were scored as apoptotic based on morphological criteria and counted. A minimum of 250 cells were counted for each condition.

Measurement of caspase activity

HK cells treated with sFasL with or without 2 µg/ml CHX for the indicated time periods were lysed in buffer containing 50 mM Tris (pH 7.0), 2 mM EDTA, and 1% Triton X-100. Cell lysates were obtained after centrifugation at 15,000 rpm. Ten micrograms of the cell lysates were incubated with 25 µM of caspase substrate, acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin (Ac-DEVD-AFC; Calbiochem) for 1 h at 37°C in the presence of caspase reaction buffer containing 100 mM HEPES (pH 7.4), 10% sucrose, 5 mM DTT, and 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Proteolysis of the fluorescent peptides was measured with excitation at 400 nm and emission at 505 nm using a fluorescence spectrophotometer.

Flow cytometry

HK cells cultured in 100-mm dishes were given TNF stimulation for 24 h. Cells were trypsinized, washed with PBS, incubated with 2 µg/ml FITC-conjugated anti-human mouse monoclonal Fas Ab (clone DX2; BD PharMingen) for 1 h at 4°C, and washed twice with PBS. The cells were then analyzed on the FACSCalibur (BD Biosciences, Mountain View, CA). The expression of surface Fas was calculated as the mean fluorescence intensity with the CellQuest program (BD Biosciences). Negative control cells were incubated with isotype-matched Ab.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
An FDC line, HK cells proliferate in response to TNF

Because TNF is mitogenic to several established cell lines and it activates ERK1/2, a molecular hallmark of proliferation in HK cells (14), we examined whether TNF acts as a growth factor in HK cells. Cells maintained in 10% FCS-containing medium were stimulated with various doses of TNF for 24 h. The effect of TNF on the proliferation of HK cells was measured by [3H]thymidine incorporation during the last 16 h chasing period. Fig. 1 shows that TNF increased the [3H]thymidine uptake dose-dependently, suggesting that TNF is mitogenic to this cell line.



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  		FIGURE 1. Effect of TNF on proliferation of HK cells. HK cells were seeded in a 96-well plate at ~5,000 cells/well. After a 24-h incubation, the cells were treated with TNF at indicated concentrations. The cells were pulsed with 0.5 µCi [3H]thymidine during the last 16-h period, and harvested onto a filter. The incorporated [3H]thymidine in DNA was measured by a liquid scintillation counter.

 
A prolonged biphasic NF-{kappa}B activation by TNF in HK cells

Most of the immunoregulatory functions of TNF are ascribed to its activity to induce a transcription factor NF-{kappa}B (20). The prototype of NF-{kappa}B is composed of two subunits (p65/RelA and p50), and is normally sequestered within the cytoplasm due to interaction with I{kappa}B{alpha}, an inhibitor of {kappa}B protein. TNF stimulation causes phosphorylation and subsequent degradation of I{kappa}B{alpha}, permitting NF-{kappa}B translocation to the nucleus, where the NF-{kappa}B transcriptionally activates a variety of genes possessing NF-{kappa}B binding elements in their promoters. Among NF-{kappa}B inducible genes is its own inhibitor I{kappa}B{alpha}, and the newly synthesized I{kappa}B{alpha} quickly interacts with NF-{kappa}B and brings it back to the cytoplasm, thereby turning off the transcription of NF-{kappa}B-driven genes (21, 22, 23). This autoregulatory mode of NF-{kappa}B activation can be observed in most cells exposed to TNF. To study the pattern of NF-{kappa}B activation by TNF in HK cells, we performed the EMSA which measures the amount of activated NF-{kappa}B bound to the appropriate NF-{kappa}B element. An NF-{kappa}B binding element that exists in the promoter of the c-IAP2 gene was used for the preparation of radiolabeled probe for this assay (19). As shown in Fig. 2A, the TNF-induced NF-{kappa}B DNA binding activity of nuclear extracts from HK cells was evident after 30-min exposure and reached maximum at 6 h after TNF treatment. Upon further incubation of cells in the presence of TNF, the NF-{kappa}B binding activity decreased slightly but not to the basal level by 24 h. After then, NF-{kappa}B activity gradually increased again until 72 h to the level seen in the cells exposed to TNF for 6 h. Similar response was also observed in another FDC-like cell line (designated as HK line 2) established from a different donor, which has been characterized by Lee et al. (15), indicating that this unique response is not only observed in a particular HK cell line (Fig. 2A, bottom panel). This biphasic prolonged mode of NF-{kappa}B activation in HK cell lines differed from the classical pattern which shows a rapid and transient mode of NF-{kappa}B activation, reaching maximum at 30 min, and then decreasing completely within 2 h.



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  		FIGURE 2. Persistent NF-{kappa}B activation and nuclear localization of p65/RelA in TNF-treated HK cells. A, Nuclear extracts of HK cells treated with 20 ng/ml TNF for the indicated times were prepared, and EMSA was performed as described in Materials and Methods using a {kappa}B probe derived from the c-IAP2 promoter (top panel). EMSA done with the HK cell line 2 from a different donor is shown (bottom panel). B, HK cells were plated on gelatin-coated coverslips and treated with TNF (20 ng/ml) for the indicated times. Cells were washed, fixed, permeabilized, and incubated with Ab to p65/RelA. Rhodamine-conjugated mouse IgG secondary Ab was used for detection. Cells were washed, and the coverslips were mounted on microscope slides and examined by fluorescence microscopy.

 
We next examined the subcellular localization of NF-{kappa}B in HK cells exposed to TNF (Fig. 2B). Cells exposed to TNF for the times shown were stained for p65/RelA and examined by fluorescence microscopy. p65/RelA was present in the cytoplasm of untreated HK cells or LF-1 fibroblasts. Both cells treated with TNF for 30 min showed p65/RelA exclusively in the nucleus. However upon further treatment for 24 h, p65/RelA was not detectable in the nucleus of LF-1 cells, while it was still found in the nucleus of HK cells, confirming the prolonged NF-{kappa}B activation.

Persistent TNF signaling in HK cells

To understand the mechanism of the sustained NF-{kappa}B activation by TNF in HK cells, we analyzed the expression dynamics of I{kappa}B{alpha} and its phosphorylation status throughout the time periods of TNF stimulation, and compared with those of LF-1 cells (Fig. 3). Thirty-minute exposure to TNF led to the complete disappearance of I{kappa}B{alpha} protein in both HK and LF-1 cells. Further incubation of LF-1 cells with TNF resulted in a complete restoration of I{kappa}B{alpha} protein. However, in HK cells I{kappa}B{alpha} reappeared but remained below at the level seen in unstimulated cells. In addition, examination of the level of phosphorylated I{kappa}B{alpha} protein revealed that TNF induced the phosphorylation of I{kappa}B{alpha} within 5 min in both cell types. In LF-1 cells that were given longer treatment of TNF, the phosphorylated I{kappa}B{alpha} was not detectable due to rapid degradation of the phosphorylated protein by the ubiquitin-proteasome pathway (20, 21, 22). In contrast, low levels of the phosphorylated I{kappa}B{alpha} species remained in HK cells throughout time periods after 30 min of TNF stimulation. These results strongly suggest that the newly synthesized I{kappa}B{alpha} is continuously subjected to phosphorylation and degradation as a result of TNF action, indicating persistent TNF signaling in HK cells.



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  		FIGURE 3. I{kappa}B{alpha} phosphorylation, degradation, and reappearance in HK or LF-1 cells treated with TNF. HK or LF-1 cells were treated for the indicated times with TNF (20 ng/ml). At the end of the treatment period, cell lysates were prepared and resolved by 10% SDS-PAGE, transferred to membranes, and immunoblotted with an anti-I{kappa}B{alpha} Ab (top panel) or with an anti-phospho-I{kappa}B{alpha} Ab (bottom panel). Blots were incubated with goat anti-rabbit IgG-conjugated HRP, and bands were visualized by ECL.

 
Persistent TNF signaling results in a prolonged biphasic induction of antiapoptotic genes, conferring resistance to TNF-induced apoptosis

To investigate whether the prolonged biphasic activation of NF-{kappa}B by TNF results in a similar mode of induction of NF-{kappa}B-inducible genes, total RNA samples were isolated from HK cells treated with TNF for time periods ranging from 2 to 72 h, and RPAs were performed using a human apoptosis multiprobe template set. As shown in Fig. 4A, TNF induced mRNAs for TRAF1, 2, 3, and 4 and mRNAs for c-IAP1 and c-IAP2. The biphasic induction kinetics of these mRNAs by TNF correlated with that of NF-{kappa}B activation, showing the first induction peak at 12–18 h and second rise in induction by 72 h of TNF treatment. The induction of TRAF1 and c-IAP2 by TNF was most prominent among the induced genes. TRAF1 and TRAF2 proteins are known to function as adaptor molecules conveying TNFR-mediated signals (24). Both c-IAP1 and c-IAP2 are also associated with TNFRs (25), and they exhibit antiapoptotic function by directly binding and inhibiting cell death protease caspases-3, -7, and -9 (26, 27). Furthermore, a recent study has suggested that these NF-{kappa}B-inducible proteins c-IAP1, c-IAP2, TRAF1, and TRAF2 all act in concert to prevent TNF-induced apoptosis at the level of caspase-8, representing an anti-apoptotic function for NF-{kappa}B (28). In fact, we observed that TNF pretreatment for 24 h rendered HK cells resistant to apoptosis induced by subsequent cotreatment with TNF and protein synthesis inhibitor CHX (Fig. 4B).



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  		FIGURE 4. The induction of antiapoptotic genes by TNF and protection of TNF-mediated apoptosis. A, Total RNA was prepared from HK cells treated with TNF for the indicated time periods and used for multiprobe RPA with the antiapoptotic gene probe set hAPO-5 (BD PharMingen). The name of each protected mRNA signal is denoted in the figure. B, HK cells either left untreated or pretreated with TNF (20 ng/ml) were washed twice with PBS, replenished with fresh medium containing TNF (20 ng/ml) and CHX (5 µg/ml), and incubated for the indicated time periods. At the end of the treatment period, cells were fixed, stained with Hoescht dye and observed with fluorescence microscopy. Cells with condensed nuclear morphology were counted and calculated for the determination of percentage of apoptosis. The representative result of three independent experiments is shown.

 
TNF enhances Fas-mediated apoptosis of HK cells

Fas (CD95), a member of the TNFR superfamily, is recognized as the principal cell surface receptor triggering apoptotic signals in a variety of cells (29), and the outcome of Fas stimulation can be influenced by a certain combination of cytokines such as TNF, IFN-{gamma}, or TGF-{beta} (30, 31, 32, 33, 34, 35, 36, 37). Although previous reports have suggested that NF-{kappa}B plays no role in the protection of Fas-mediated apoptosis (28, 38), some recent evidence has emerged showing the protective role of NF-{kappa}B in Fas-mediated apoptosis of T lymphocytes (39) and leukemic eosinophils (40). Hence, we investigated whether HK cells can die by Fas activation and their susceptibility is regulated by TNF-induced NF-{kappa}B (Fig. 5). Sixteen hours of treatment of HK cells with sFasL alone led to 21% of apoptosis, and together with CHX, led to 62% of apoptosis, indicating that HK cells are sensitive to Fas ligation. Furthermore, their Fas sensitivity was shown to increase in 24-h TNF-pretreated cells, which upon stimulation with sFasL, led to increased apoptosis of 42% and together with CHX, led to 92% (Fig. 5A, {blacksquare}).



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  		FIGURE 5. TNF sensitization of HK cells to Fas-mediated apoptosis. Unprimed HK cells or cells pretreated with TNF (20 ng/ml) for 24 h were washed twice with PBS, and prepared for treatment with sFasL alone or with sFasL plus CHX (2 µg/ml) in fresh complete medium. A, After 16 h exposure of cells to the death-inducing stimuli, cells were fixed, stained with Hoescht dye and counted for apoptosis. The data is representative of five independent experiments. B, Total clear lysates were prepared from the cells (-, unprimed; +, 24-h TNF-pretreated) exposed to CHX (2 µg/ml), sFasL, or sFasL plus CHX for 12 h, and cleavage of caspase-8, caspase-3, and PARP was examined by Western blot analysis using the respective Abs. C, Unprimed or TNF-primed HK cells were stimulated with sFasL in the presence of CHX for the various time periods and processed for determining caspase-3 activity. Cell lysates were incubated with the fluorogenic caspase substrate Ac-DEVD-AFC and free AFC was measured at an excitation wavelength of 400 nm and an emission wavelength of 505 nm.

 
The enhanced Fas-mediated cell death by TNF was further confirmed by increased production of the cleaved fragments of caspase-3 and -8, those generated as a consequence of Fas activation (Fig. 5B). Western blot analysis using polyclonal caspase-3 and -8 Abs showed that, upon sFasL treatment of unprimed HK cells in the presence of CHX, procaspase-8 was cleaved into products with molecular masses of 43, 41, and 23 kDa; likewise, caspase-3 was cleaved into 17- and 19-kDa products. The amount of the cleaved products was increased in TNF-pretreated cells in response to sFasL treatment. To determine whether TNF-pretreated cells have increased caspase-3 activity induced by sFasL, we measured this enzyme activity by using an in vitro fluorogenic substrate DEVD-AFC (Fig. 5C) and investigated the cleavage of PARP, a cellular substrate of caspase-3 (Fig. 5B). Both assays confirmed that TNF pretreatment increased the activation of the distal caspase-3 by sFasL treatment. Collectively, these results indicate that TNF sensitizes HK cells toward Fas-mediated apoptosis, wherein TNF-induced antiapoptotic proteins appear to have no protective role.

The increased susceptibility to Fas killing by TNF is caused by up-regulating surface Fas expression via NF-{kappa}B

Because increased Fas susceptibility by TNF in many cell types is due to the up-regulation of Fas (30, 32, 33, 34, 35), we analyzed the surface Fas level of HK cells after TNF treatment (Fig. 6). Whereas untreated HK cells expressed low level of Fas, treatment of the cells with TNF for 24 h led to significant up-regulation of surface Fas. The number of cells with fluorescence value above the untreated control increased after stimulation with TNF by 2.1-fold. Western blot analysis was performed to confirm the result of flow cytometry (Fig. 6B). Ab raised against the cytoplasmic domain of Fas detected three main forms of Fas. Among these, TNF increased the level of a high molecular mass of Fas in a time-dependent manner. Heterogeneous immunoreactive bands with this Ab were observed in other cell types including Jurkat T cells, Ramos B cells, and HeLa cells. A recent report has shown that like HK cells, the increased level of the Fas species with high molecular mass by TNF and/or IFN-{gamma} correlates with the increased susceptibility to Fas-mediated cell death in thyroid follicular cells (30).



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  		FIGURE 6. TNF up-regulation of surface Fas expression on HK cells. A, HK cells were treated with 20 ng/ml TNF for 24 h. Cells were then trypsinized for analysis of surface Fas expression by immunofluorescence flow cytometry. Cells treated with TNF are shown in the shaded area and untreated cells in black open line. Cells stained with isotype-matched control Ab are indicated by the dotted line. B, Total cell lysates of HK cells treated with TNF for the indicated times were analyzed by Western blot for Fas expression (left panel). Western blot analysis of total cell lysates from other cell types was performed to examine the heterogenous forms of Fas (right panel).

 
It has been known that NF-{kappa}B is implicated in the inducible expression of Fas by various activation routes (41, 42, 43), and its contribution to the TNF-induced Fas expression has been reported in mouse fibroblasts (42) and microglial cells (32). To find out whether NF-{kappa}B activation by TNF is responsible for up-regulation of Fas, HK cells were pretreated with the well-established NF-{kappa}B inhibitor, ALLN for 30 min. After TNF stimulation, total RNA samples were prepared for Northern blot analysis. Fig. 7A shows that TNF treatment led to the induction of Fas mRNA, and pretreament of ALLN inhibited TNF-dependent Fas induction, indicating that Fas is a target of TNF-induced NF-{kappa}B activation in this cell line. When the surface Fas level was examined in HK cell line 2 by FACS analysis, TNF up-regulation of Fas expression was blocked by ALLN (Fig. 7B). Seeking further evidence for the contribution of NF-{kappa}B to induction of Fas, HK cells were infected with recombinant adenovirus expressing I{kappa}B{alpha} super-repressor to block TNF-induced NF-{kappa}B activation (17). After treating cells with TNF for the indicated times, lysates were prepared and processed for Western blot analysis. As shown in Fig. 7B, cells infected with virus expressing the I{kappa}B{alpha} super-repressor did not show TNF-induced increase in the level of high molecular mass species of Fas protein, while TNF-induced up-regulation of this species was clearly visible in cells that were uninfected or infected with control virus expressing {beta}-galactosidase. Collectively, these results indicate that increased Fas susceptibility by TNF can be attributed to its NF-{kappa}B-inducing activity that leads to up-regulation of surface Fas expression on HK cells.



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  		FIGURE 7. NF-{kappa}B contributes to TNF-inducible Fas expression on HK cells. A, HK cells were treated with DMSO for negative control (-) or with NF-{kappa}B inhibitor, ALLN (20 µM) for 30 min before TNF stimulation. After 6 and 18 h of stimulation, total RNA was extracted and analyzed by Northern blotting using a radiolabeled Fas cDNA probe. The indicated 28S and 18S rRNAs ensure the equal loading of the RNA samples. B, Confluent cultures of HK cell line 2 were left untreated or treated with ALLN before TNF stimulation. Twenty-four hours later, cells were analyzed for surface Fas expression using FACS. The upper histogram shaded with light gray is obtained from untreated control. In the bottom panel, the solid-line histogram is obtained from cells treated with TNF for 24 h and the histogram shaded with dark gray is from cells treated sequentially with ALLN for 30 min and then with TNF for 24 h. C, HK cells infected with adenovirus expressing flag-tagged I{kappa}B{alpha} super-repressor (Ad I{kappa}B{alpha} super-repressor) or {beta}-galactosidase (Ad {beta}-Gal) at the multiplicity of infection of 100 are indicated by +. After 48 h infection, the virus infected cells (+) and uninfected cells (-) were treated with TNF for the indicated times. Total cell lysates were prepared and examined with Western blot analysis for Fas expression (top panel). Expression of I{kappa}B{alpha} super-repressor in infected cells was confirmed by Western blot analysis with Ab against I{kappa}B{alpha} (bottom panel).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies with gene-targeted mice have revealed essential roles for TNF and its receptor in the formation of FDC networks and GC in the secondary lymphoid organs (7, 8, 9, 10). Despite such well-recognized in vivo roles of TNF, information about the in vitro effects of this cytokine on FDC function is limited. In the present study, we report that an established FDC line of HK cells proliferated in response to TNF and displayed a distinct persistent NF-{kappa}B activation mode, resulting in a sustained induction of NF-{kappa}B-inducible genes. In addition, the TNF-activated NF-{kappa}B is shown to contribute to the enhancement of surface Fas expression on HK cells, thereby rendering the cells more susceptible to Fas-induced apoptosis.

FDCs mature from precursor cells and constitute the GC framework by their elongated cytoplasmic processes, conglomerating with GC B cells. The life cycle of FDC in the lymphoid follicles is largely unknown. It is considered that FDCs do not proliferate, or are slowly turned over (44, 45). However, it is reasonable to assume that FDC precursor cells precasted in the primary B cell follicles should proliferate and differentiate to form FDC networks upon Ag stimulation. In fact, in normal mice, specific blockage of TNF signals for 3 wk caused the disappearance of pre-existing FDC networks in the primary follicles of nonimmunized mice (46, 47, 48). However, this effect was not observed in the secondary follicles of immunized mice, suggesting that a function of TNF is restricted to the maintenance of a pre-existing FDC network (48). Other experiments using wild-type mice reconstituted with bone marrow lymphocytes of TNF knockout mice have shown that more prolonged TNF deficiency for longer than 10 wk was required to observe the defect in the formation of FDC networks upon Ag challenge, speculating that population of FDCs may not be replenished with newly generated precursors under long-term absence of TNF signals (49). In this context, it is conceivable that an important function of TNF in the induction and maintenance of a FDC network relies on the mitogenic action to the FDC.

Another important function of TNF can be attributable to its NF-{kappa}B-inducing activity which leads to enhanced cytokine and chemokine production and adhesion molecule expression such as VCAM-1 and ICAM-1, all that provide potentially key signals to support GC B cell survival and differentiation (6, 50). Our present data show that the TNF-induced NF-{kappa}B activation pattern observed in two independently established HK cell lines is quite different from other cells. HK cells do not follow the self-regulating mode of NF-{kappa}B activation which exhibits rapid and transient activation of NF-{kappa}B. Instead, they showed a sustained biphasic NF-{kappa}B activation, as evidenced by prolonged NF-{kappa}B DNA binding activity up to 72 h and persistent nuclear localization of p65/RelA (Fig. 2). Analysis of the mechanism for the prolonged NF-{kappa}B activation revealed that HK cells do not become desensitized by TNF signaling. This was shown by the incomplete resynthesis of I{kappa}B{alpha} protein and the presence of a low level of phosphorylated I{kappa}B{alpha} throughout the time periods of TNF exposure, which suggest a steady state resynthesis, phosphorylation, and subsequent degradation of I{kappa}B{alpha} (Fig. 3). Persistent NF-{kappa}B activation by TNF has been reported in normal foreskin fibroblasts and its physiological relevance in many chronic inflammatory diseases has been proposed (51). It is likely that the persistency of NF-{kappa}B activity in FDC may play a role in perpetuating a cycle of cellular activation and the guided localization of FDC and lymphocytes to proper compartments in the secondary lymphoid follicles through stable expression of adhesion molecules, cytokines, and chemokines.

NF-{kappa}B plays a protective role in TNF-induced apoptosis by inducing genes that inhibit cell death (28, 38). Persistent TNF signaling in HK cells led to persistent induction of antiapoptotic genes (Fig. 4). At the same time, our data demonstrated that TNF pretreatment potentiated sFasL-induced apoptosis by an NF-{kappa}B-dependent increase in surface Fas expression. This increased susceptibility of Fas killing in the FDC line by TNF seems paradoxical in view of the important role of TNF in establishment of FDC networks which presumably depends on its mitogenic action on putative FDC precursors. However, the consequence induced by Fas activation on FDCs needs to be considered in the context of the biological source of FasL in GC microenvironments. Although the presence of FasL in the GCs is controversial, several reports have suggested that FasL is expressed in the GC (52, 53, 54, 55). Especially, Verbeke et al. (55) detected FasL proteins by Western blot analysis and immunohistochemistry in human tonsils and lymph nodes obtained from patients with lymphofollicular hyperplasia. Furthermore, they showed that the expression of FasL is confined to the FDC-associated cell clusters in the light zone of the GC, speculating that FDCs express FasL. Although they do not point out whether FasL induces FDC death, the results suggest that, under certain circumstances, FDCs may be subjected to Fas activation. FasL is expressed in most of the tumor tissues from Hodgkin lymphoma patients in which FDC networks are severely disrupted (56). In addition, in lymph nodes of patients infected with HIV, there is destruction of FDC networks and invasion of CD8-positive T cells into the GC (57, 58, 59, 60). Although the mechanism by which FDC networks are destroyed in these pathological conditions is unknown, these FasL-bearing lymphocytes themselves or their shedded products (sFasL) may, at least, in part contribute to the destruction of FDC networks.

In addition to the presence of FasL, other factors present in the GC microenvironment may influence the induction of the final consequences of Fas activation on FDC. Given the prolonged survival of FDC networks in the secondary lymphoid follicles, FDCs participating actively in the GC reaction may not be prone to death by apoptosis despite the presence of FasL. There may exist factors in the GC microenvironment that maintain FDC networks by sending signals that counteract TNF action and inhibit Fas activation on FDC. A possible candidate playing such role is the cytokine TGF-{beta}, because it is expressed in the GC by both FDCs and lymphocytes (61), and is known as a potent modulator of Fas-mediated apoptosis in a variety of cell types (32, 36, 37). Our experiments in progress have revealed that TGF-{beta} indeed inhibits Fas-mediated apoptosis of HK cells (S.-M. Park and T. H. Lee, unpublished data).

In summary, the ultimate fate of FDC following Fas activation is the result of a complex interplay between signals that are delivered by environmental factors at the time when Fas is engaged. Sensitivity to Fas can be regulated by the activation or differentiation state of the cells, which affects the ability of the cells to receive and process death-inducing signal. A study evaluating the FDC network for a long period of time after induction of GC responses reveals that the FDC networks degenerate in the resolution stage of GC, but occasional small "islands" of tightly packed Ag-bearing FDC are identifiable for months to years after Ag clearance (62). How these long-lasting FDC networks are maintained even after clearance of most of the Ag, and whether the major population of FDC in the involuting follicles of normal lymphoid tissues undergoes programmed cell death are subjects of open question. Our results suggest that FDCs in vivo undergo apoptotic cell death through Fas activation, and the process can be controlled by TNF and/or other cytokines present in the GC microenvironment in a positive or negative manner, depending on the differentiation stages of FDC maturation.


   Footnotes
 
1 This work was supported by Ministry of Health and Welfare Grant HMP-00-B-20700-0019 and by a grant from the Protein Network Research Center (Yonsei University), the Ministry of Science and Technology/Korea Science and Engineering Foundation. Back

2 Address correspondence and reprint requests to Dr. Tae H. Lee, Department of Biology, College of Science, Yonsei University, 134 Shinchon, Seodaemoon, Seoul 120-749, Korea. E-mail address: thlee{at}yonsei.ac.kr Back

3 Abbreviations used in this paper: GC, germinal center; FDC, follicular dendritic cell; ERK, extracellular signal-regulated kinase; TRAF, TNFR-associated factor; c-IAP, cellular inhibitor of apoptosis protein; sFasL, soluble Fas ligand; RPA, RNase protection assay; CHX, cycloheximide; PARP, poly(ADP-ribose) polymerase. Back

Received for publication February 25, 2003. Accepted for publication August 6, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kelsoe, G.. 1995. The germinal center reaction. Immunol. Today 16:324.[Medline]
  2. MacLennan, I. C. M.. 1994. Germinal centers. Annu. Rev. Immunol. 12:117.[Medline]
  3. Billian, G., P. Mondière, M. Berard, C. Bella, T. Defrance. 1997. Antigen receptor-induced apoptosis of human germinal center B cells is targeted to a centrocytic subset. Eur. J. Immunol. 27:405.[Medline]
  4. Kosco, M. H., E. Pflugfelder, D. Gray. 1992. Follicular dendritic cell-dependent adhesion and proliferation of B cells in vitro. J. Immunol. 148:2331.[Abstract]
  5. Tew, J. G., R. M. Dilosa, G. F. Burton, M. H. Kosco, L. I. Kupp, A. Masuda, A. K. Szakal. 1992. Germinal centers and antibody production in bone marrow. Immunol. Rev. 126:99.[Medline]
  6. Koopman, G., R. M. J. Keehnen, E. Lindhout, W. Newman, Y. Shimizu, G. A. van Seventer, C. de Groot, S. T. Pals. 1994. Adhesion through the LFA-1 (CD11a/CD18)-ICAM-1 (CD54) and the VLA-4 (CD49d)-VCAM-1 (CD106) pathways prevents apoptosis of germinal center B cells. J. Immunol. 152:3760.[Abstract]
  7. Pasparakis, M., L. Alexopoulou, V. Episkopou, G. Kollias. 1996. Immune and inflammatory responses in TNF{alpha}-deficient mice: a critical requirement for TNF{alpha} in the formation of primary B cell follicles, follicular dendritic cell networks and germinal centers, and in the maturation of the humoral immune response. J. Exp. Med. 184:1397.[Abstract/Free Full Text]
  8. Pasparakis, M., L. Alexopoulou, M. Grell, K. Pfizenmaier, H. Bluethmann, G. Kollias. 1997. Peyer’s patch organogenesis is intact yet formation of B lymphocyte follicles is defective in peripheral lymphoid organs of mice deficient for tumor necrosis factor and its 55-kDa receptor. Proc. Natl. Acad. Sci. USA 94:6319.[Abstract/Free Full Text]
  9. Tkachuk, M., S. Bolliger, B. Ryffel, G. Pluschke, T. A. Banks, S. Herren, R. H. Gisler, M. H. Kosco-Vilbois. 1998. Crucial role of tumor necrosis factor receptor 1 expression on nonhematopoietic cells for B cell localization within the splenic white pulp. J. Exp. Med. 187:469.[Abstract/Free Full Text]
  10. Matsumoto, M., Y. X. Fu, H. Molina, G. Huang, J. Kim, D. A. Thomas, M. H. Nahm, D. D. Chaplin. 1997. Distinct roles of lymphotoxin and the type I tumor necrosis factor (TNF) receptor in the establishment of follicular dendritic cells from non-bone marrow-derived cells. J. Exp. Med. 186:1997.[Abstract/Free Full Text]
  11. Kim, H.-S., X. Zhang, Y. S. Choi. 1994. Activation and proliferation of follicular dendritic cell-like cells by activated T lymphocytes. J. Immunol. 153:2951.[Abstract]
  12. Kim, H.-S., X. Zhang, E. Klyushnenkova, Y. S. Choi. 1995. Stimulation of germinal center B lymphocyte proliferation by an FDC-like cell line, HK. J. Immunol. 155:1101.[Abstract]
  13. Choe, J., H.-S. Kim, X. Zhang, R. J. Armitage, Y. S. Choi. 1996. Cellular and molecular factors that regulate the differentiation and apoptosis of germinal center B cells: anti-Ig down-regulates Fas expression on CD40L-stimulated germinal center B cells and inhibits Fas-mediated apoptosis. J. Immunol. 157:1006.[Abstract]
  14. Park, S.-M., H.-S. Kim, J. Choe, T. H. Lee. 1999. Differential induction of cytokine genes and activation of mitogen-activated protein kinase family and soluble CD-40 ligand and TNF in a human follicular dendritic cell line. J. Immunol. 163:631.[Abstract/Free Full Text]
  15. Lee, I. Y., J. Choe. 2003. Human follicular dendritic cells and fibroblasts share the 3C8 antigen. Biochem. Biophys. Res. Commun. 304:701.[Medline]
  16. Ahn, J. H., S. M. Park, H. S. Cho, M. S. Lee, J. B. Yoon, J. Vilcek, T. H. Lee. 2001. Non-apoptotic signaling pathways activated by soluble Fas ligand in serum-starved human fibroblasts: mitogen-activated protein kinases and NF-{kappa}B-dependent gene expression. J. Biol. Chem. 276:47100.[Abstract/Free Full Text]
  17. Kim, J. Y., S. Lee, B. Hwangbo, C. T. Lee, Y. W. Kim, S. K. Han, Y. S. Shim, C. G. Yoo. 2000. NF-{kappa}B activation is related to the resistance of lung cancer cells to TNF-{alpha}-induced apoptosis. Biochem. Biophys. Res. Commun. 273:140.[Medline]
  18. Dignam, J. D., R. M. Lebovitz, R. G. Roeder. 1983. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11:1475.[Abstract/Free Full Text]
  19. Hong, S.-Y., W.-H. Yoon, J.-H. Park, S.-G. Kang, J.-H. Ahn, T. H. Lee. 2000. Involvement of two NF-{kappa}B binding elements in tumor necrosis factor {alpha}-, CD40-, and Epstein-Barr virus latent membrane protein 1-mediated induction of the cellular inhibitor of apoptosis protein 2 gene. J. Biol. Chem. 275:10822.
  20. Baeuerle, P. A., T. Henkel. 1994. Function and activation of NF-{kappa}B in the immune system. Annu. Rev. Immunol. 12:141.[Medline]
  21. Ghosh, S., M. J. May, E. B. Kopp. 1998. NF-{kappa}B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu. Rev. Immunol. 16:225.[Medline]
  22. Karin, M.. 1999. How NF-{kappa}B is activated: the role of the I{kappa}B kinase (IKK) complex. Oncogene 18:6867.[Medline]
  23. Pahl, H. L.. 1999. Activators and target genes of Rel/NF-{kappa}B transcription factors. Oncogene 18:6853.[Medline]
  24. Rothe, M., V. Sarma, V. M. Dixit, D. V. Goeddel. 1995. TRAF2-mediated activation of NF-{kappa}B by TNF receptor 2 and CD40. Science 269:1424.[Abstract/Free Full Text]
  25. Rothe, M., M. G. Pan, W. J. Henzel, T. M. Ayres, D. V. Goeddel. 1995. The TNFR2-TRAF signaling complex contains two novel proteins related to baculoviral inhibitor of apoptosis proteins. Cell 83:1243.[Medline]
  26. Roy, N., Q. L. Deveraux, R. Takahashi, G. S. Salvesen, J. C. Reed. 1997. The c-IAP-1 and c-IAP-2 proteins are direct inhibitors of specific caspases. EMBO J. 16:6914.[Medline]
  27. Deveraux, Q. L., N. Roy, H. R. Stennicke, T. Van Arsdale, Q. Zhou, S. M. Srinivasula, E. S. Alnemri, G. S. Salvesen, J. C. Reed. 1998. IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases. EMBO J. 17:2215.[Medline]
  28. Wang, C. Y., M. W. Mayo, R. G. Korneluk, D. V. Goeddel, A. S. Baldwin, Jr. 1998. NF-{kappa}B antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science 281:1680.[Abstract/Free Full Text]
  29. Nagata, S.. 1999. Fas ligand-induced apoptosis. Annu. Rev. Genet. 33:29.[Medline]
  30. Bretz, J. D., P. L. Arscott, A. Myc, J. R. Barker, Jr. 1999. Inflammatory cytokine regulation of Fas-mediated apoptosis in thyroid follicular cells. J. Biol. Chem. 274:25433.[Abstract/Free Full Text]
  31. Kobayashi, T., K. Okamoto, T. Kobata, T. Hasunuma, T. Kato, H. Hamada, K. Nishioka. 2000. Differential regulation of Fas-mediated apoptosis of rheumatoid synoviocytes by tumor necrosis factor {alpha} and basic fibroblast growth factor is associated with the expression of apoptosis-related molecules. Arthritis Rheum. 43:1106.[Medline]
  32. Lee, S. J., T. Zhou, C. Choi, Z. Wang, E. N. Benveniste. 2000. Differential regulation and function of Fas expression on glial cells. J. Immunol. 164:1277.[Abstract/Free Full Text]
  33. Spanaus, K. S., R. Schlapbach, A. Fontana. 1998. TNF-{alpha} and IFN-{gamma} render microglia sensitive to Fas ligand-induced apoptosis by induction of Fas expression and down-regulation of Bcl-2 and Bcl-xL. Eur. J. Immunol. 28:4398.[Medline]
  34. Riccioli, A., D. Starace, A. D’Alessio, G. Starace, F. Padula, P. De Cesaris, A. Filippini, E. Ziparo. 2000. TNF-{alpha} and IFN-{gamma} regulate expression and function of the Fas system in the seminiferous epithelium. J. Immunol. 165:743.[Abstract/Free Full Text]
  35. Luttmann, W., A. Opfer, E. Dauer, M. Foerster, H. Matthys, H. Eibel, K. Schulze-Osthoff, C. Kroegel, J. C. Virchow. 1998. Differential regulation of CD95 (Fas/APO-1) expression in human blood eosinophils. Eur. J. Immunol. 28:2057.[Medline]
  36. Schlapbach, R., K. S. Spanaus, U. Malipiero, S. Lens, A. Tasinato, J. Tschopp, A. Fontana. 2000. TGF-{beta} induces the expression of the FLICE-inhibitory protein and inhibits Fas-mediated apoptosis of microglia. Eur. J. Immunol. 30:3680.[Medline]
  37. Dybedal, I., F. Guan, O. J. Borge, O. P. Veiby, V. Ramsfjell, S. Nagata, S. E. Jacobsen. 1997. Transforming growth factor-{beta}1 abrogates Fas-induced growth suppression and apoptosis of murine bone marrow progenitor cells. Blood 90:3395.[Abstract/Free Full Text]
  38. Van Antwerp, D. J., S. J. Martin, T. Kafri, D. R. Green, I. M. Verma. 1997. Suppression of TNF-{alpha}-induced apoptosis by NF-{kappa}B. Science 274:787.
  39. Dudley, E., F. Hornung, L. Zheng, D. Scherer, D. Ballard, M. Lenardo. 1999. NF-{kappa}B regulates Fas/APO-1/CD95- and TCR-mediated apoptosis of T lymphocytes. Eur. J. Immunol. 29:878.[Medline]
  40. Qin, Y., B. Camoretti-Mercado, L. Blokh, C. G. Long, F. D. Ko, K. J. Hamann. 2002. Fas resistance of leukemic eosinophils is due to activation of NF-{kappa}B by Fas ligation. J. Immunol. 169:3536.[Abstract/Free Full Text]
  41. Ouaaz, F., M. Li, A. A. Beg. 1999. A critical role for the RelA subunit of nuclear factor {kappa}B in regulation of multiple immune-response genes and in Fas-induced cell death. J. Exp. Med. 189:999.[Abstract/Free Full Text]
  42. Zheng, Y., F. Ouaaz, P. Bruzzo, V. Singh, S. Gerondakis, A. A. Beg. 2001. NF-{kappa}B RelA (p65) is essential for TNF-{alpha}-induced Fas expression but dispensable for both TCR-induced expression and activation-induced cell death. J. Immunol. 166:4949.[Abstract/Free Full Text]
  43. Chan, H., D. P. Bartos, L. B. Owen-Schaub. 1999. Activation-dependent transcriptional regulation of the human Fas promoter requires NF-{kappa}B p50–p65 recruitment. Mol. Cell. Biol. 19:2098.[Abstract/Free Full Text]
  44. Kapasi, Z. F., D. Qin, W. G. Kerr, M. H. Kosco-Vilbois, L. D. Shultz, J. G. Tew, A. K. Szakal. 1998. Follicular dendritic cell (FDC) precursors in primary lymphoid tissues. J. Immunol. 160:1078.[Abstract/Free Full Text]
  45. Endres, R., M. B. Alimzhanov, T. Plitz, A. Futterer, V. M. Kosco, S. A. Nedospasov, K. Rajewsky, K. Pfeffer. 1999. Mature follicular dendritic cell networks depend on expression of lymphotoxin{beta} receptor by radioresistant stromal cells and of lymphotoxin {beta} and tumor necrosis factor by B cells. J. Exp. Med. 189:159.[Abstract/Free Full Text]
  46. Mackay, F., G. R. Majeau, P. Lawton, P. S. Hochman, J. L. Browning. 1997. Lymphotoxin but not tumor necrosis factor functions to maintain splenic architecture and humoral responsiveness in adult mice. Eur. J. Immunol. 27:2033.[Medline]
  47. Cook, M. C., H. Korner, D. S. Riminton, F. A. Lemckert, J. Hasbold, M. Amesbury, P. D. Hodgkin, J. G. Cyster, J. D. Sedgwick, A. Basten. 1998. Generation of splenic follicular structure and B cell movement in tumor necrosis factor-deficient mice. J. Exp. Med. 188:1503.[Abstract/Free Full Text]
  48. Mackay, F., J. L. Browning. 1998. Turning off follicular dendritic cells. Nature 395:26.[Medline]
  49. Wang, Y., J. Wang, Y. Sun, Q. Wu, Y. X. Fu. 2001. Complementary effects of TNF and lymphotoxin on the formation of germinal center and follicular dendritic cells. J. Immunol. 166:330.[Abstract/Free Full Text]
  50. Husson, H., S. M. Lugli, P. Ghia, A. Cardoso, A. Roth, K. Brohmi, E. G. Carideo, Y. S. Choi, J. Browning, A. S. Freedman. 2000. Functional effects of TNF and lymphotoxin {alpha}1{beta}2 on FDC-like cells. Cell. Immunol. 203:134.[Medline]
  51. Poppers, D. M., P. Schwenger, J. Vilcek. 2000. Persistent tumor necrosis factor signaling in normal human fibroblasts prevents the complete resynthesis of I{kappa}B-{alpha}. J. Biol. Chem. 275:29587.[Abstract/Free Full Text]
  52. Kondo, E., T. Yoshino, R. Nishiuchi, I. Sakuma, K. Nishizaki, N. Kayagaki, H. Yagita, T. Akagi. 1997. Expression of Fas ligand mRNA in germinal centres of the human tonsil. J. Pathol. 183:75.[Medline]
  53. Sträter, J., S. M. Mariani, H. Walczak, F. G. Rucker, F. Leithauser, P. H. Krammer, P. Moller. 1999. CD95 ligand (CD95L) in normal human lymphoid tissues: a subset of plasma cells are prominent producers of CD95L. Am. J. Pathol. 154:193.[Abstract/Free Full Text]
  54. Hur, D. Y., D. J. Kim, S. Kim, Y. I. Kim, D. Cho, D. S. Lee, Y. Hwang, K. Bae, K. Y. Chang, W. J. Lee. 2000. Role of follicular dendritic cells in the apoptosis of germinal center B cells. Immunol. Lett. 72:107.[Medline]
  55. Verbeke, C. S., U. Wenthe, H. Zentgraf. 1999. Fas ligand expression in the germinal centre. J. Pathol. 189:155.[Medline]
  56. Verbeke, C. S., U. Wenthe, R. Grobholz, H. Zentgraf. 2001. Fas ligand expression in Hodgkin lymphoma. Am. J. Surg. Pathol. 25:388.[Medline]
  57. Burton, G. F., B. F. Keele, J. D. Estes, T. C. Thacker, S. Gartner. 2002. Follicular dendritic cell contributions to HIV pathogenesis. Semin. Immunol. 14:275.[Medline]
  58. Mori, S., Y. Ezaki, M. Mori, M. Takahashi, M. Teshima, K. Sagawa. 1998. Deterioration of B cell proliferation correlates with dendritic reticulum cell destruction in germinal centers of an AIDS patient. Case study. Acta Pathol. Jpn. 38:1205.
  59. Piris, M. A., C. Rivas, M. Morente, C. Rubio, C. Martin, H. Olivia. 1987. Persistent and generalized lymphadenopathy: a lesion of follicular dendritic cells: an immunohistologic and ultrastructural study. Am. J. Clin. Pathol. 87:716.[Medline]
  60. Said, J. W., J. L. Pinkus, J. Yamashita, S. Mishalani, F. Matsumura, S. Yamashiro, G. S. Pinkus. 1997. The role of follicular and interdigitating dendritic cells in HIV-related lymphoid hyperplasia: localization of fascin. Mod. Pathol. 10:421.[Medline]
  61. Yamada, K., M. Yamakawa, Y. Imai, M. Tsukamoto. 1997. Expression of cytokine receptors on follicular dendritic cells. Blood 90:4832.[Abstract/Free Full Text]
  62. Kosco-Vilbois, M. H., D. Scheidegger. 1995. Follicular dendritic cells: antigen retention, B cell activation, and cytokine production. Curr. Top. Microbiol. Immunol. 201:69.[Medline]



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