Introduction
Lymph node (LN) is a starting point for immune response of immune cells. Pathogen infection leads to LN expansion and markedly increases LN cellularity [24]. Although lymphocyte proliferation and vascular remodeling contribute to this expansion, the contributions of the reticular network have not been elucidated. Recently, this swelling led to increased spacing in the paracortical reticular network and allowed LNs to expand and enlarged space within reticulum resulted in an enhanced immunity [2]. The physical plasticity of LN is maintained in part by podoplanin (PDPN) signaling in stromal fibroblastic reticular cells (FRCs) of T zone and its modulation by CLEC-2 expressed on dendritic cells [2]. PDPN induces actomyosin contractility in FRCs via activation of RhoA/C and downstream Rho-associated protein kinase (ROCK) [1]. Stress fiber (SF) is composed of actin bundle held together by α-actinin, fascin, espin, filamin and myosin II bundle [15]. They generate contractile force and play a central role in morphogenesis, motility, and adhesion of eukaryotic cells [14]. FRCs form a continuous stromal network comprising the collagen-rich reticular fibers, another mesenchymal cell population and the thin strand of extracellular matrix (ECM) components that make up the FRC conduit network [4]. FRC network stretching allows for the rapid LN expansion--driven by lymphocyte influx and proliferation--that is the critical hallmark of adaptive immunity [4]. The characteristic network made by FRCs provides mechanical strength to the tissue and makes spaces for T cell motility in LN [2]. Thus, this plasticity of FRC might be linked to alteration. Although CLEC-2–podoplanin axis contribute to this spacing [2], the spacing contributions of the lymphotoxins (LT) have not been elucidated in FRC. LTs, members of TNF family cytokines including soluble homotrimeric LTα and the membrane heterotrimeric form of LTα1 β2, are important for lymphoid tissue architecture [18]. LTα shares its receptors with TNFα (TNFRI and TNFRII) and, thus, the two factors elicit similar effects [5]. TNFα activates the NF-κB, chiefly of the RelA (p65)–p50 complex pathway [10]. In contrast, LTα1β2 activates a distinct signaling pathway toward RelB–p52 (p100) via LTβR [12]. We examined the roles of these tow mediators in the cytoskeleton alteration, morphological change and making space in FRC. Here, in order to investigate morphologic change and making space of FRCs when FRCs are treated with LT members, the cytoskeleton, with particular emphasis on associated with filamentous actin (F-actin), was studied in FRC treated with several stimulators.
Material and method
Cells
FRCs were established as described previously [9]. FRC was maintained in 10% FCS–DMEM medium supplemented with streptomycin and penicillin.
Treatment of FRCs with LTα, anti-LTβR antibody, ML-7 and TNFα
Several monoclonal antibodies and polyclonal antibody raised to the extracellular part of LTβR receptor and can trigger downstream signals [13, 17, 22]. FRC monolayers (105 cells/ml) were grown on cover slips in cell culture dishes for fluorescence microscopy. After 24 hr of incubation, 10 ng/ml LTα, 1 μg/ml anti-LTβR antibody, 5 μM ML-7 and 100 ng/ml TNFα were added. Incubation was continued for 24 hr, and the cultures were processed as described below.
Fluorescence microscopy
FRCs were treated with LTα, anti-LTβR antibody, ML-7 and TNFα as described above, were gently rinsed, fixed in 4% formaldehyde in PBS for 5 min, washed with PBS. Cells were stained with 5 μg/ml rhodamine-conjugated phalloidin to visualize F-actin for 45 min. Stained cover slips were examined with a Zeiss photomicroscope equipped for fluorescence microscopy.
Western blot
FRCs were pretreated with anti-LTβR antibody and were lysed in 5× SDS sample buffer. After the samples were boiled, equal amounts of total lysates were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were soaked in a blocking solution (5% skim milk and 0.2% Tween 20-PBS) for 1 hr, and then incubated with primary antibodies for 1 hr. After being washed with Tween 20-PBS, membranes were incubated with appropriate HRP-conjugated secondary antibodies for 1 hr. Specific bands were visualized by an ECL method (Amersham Biosciences, Piscataway, NJ, USA ).
RNA isolation
Total RNA was isolated by using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). The RNA were extracted with chloroform (Sigma Chemical, St. Louis, MO, USA) and followed by ethanol precipitation. RNA was quantified using NanoDrop® ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA). And RNA quality was assessed using Agilent RNA Nano 6000 LabChip kits and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA).
Microarray analysis
For control and test RNAs, the synthesis of target cRNA probes and hybridization were performed using Agilent’s Low RNA Input Linear Amplification kit (Agilent Technologies, Palo Alto, CA, USA) according to the manufacturer’s instructions. Briefly, each 1 μg total RNA and T7 promoter primer mix and incubated at 65℃ for 10 min. cDNA master mix (5X First strand buffer, 0.1 M DTT, 10 mM dNTP mix, RNase-Out, and MMLV-RT) was prepared and added to the reaction mixer. The samples were incubated at 40℃ for 2 hr and then the RT and dsDNA synthesis was terminated by incubating at 65℃ for 15 min. The transcription master mix was prepared as the manufacturer’s protocol (4X Transcription buffer, 0.1M DTT, NTP mix, 50% PEG, RNase-Out, Inorganic pyrophosphatase, T7-RNA polymerase, and Cyanine 3/5-CTP). Transcription of dsDNA was preformed by adding the transcription master mix to the dsDNA reaction samples and incubating at 40℃ for 2 hr. Amplified and labeled cRNA was purified on cRNA Cleanup Module according to the manufacturer’s protocol. Labeled cRNA target was quantified using ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA). After checking labeling efficiency, fragmentation of cRNA was performed by adding 10X blocking agent and 25X fragmentation buffer and incubating at 60℃ for 30 min. The fragmented cRNA was resuspended with 2X hybrid-ization buffer and directly pipetted onto assembled Agilent’s Mouse Oligo Microarray (44K). The arrays hybridized at 65℃ for 17 hr using Agilent Hybridization oven. The hybridized microarrays were washed as the manufacturer’s washing protocol.
Data acquisition and analysis
The hybridized images were scanned using Agilent’s DNA microarray scanner and quantified with Feature Extraction Software (Agilent Technology, Palo Alto, CA, USA). All data normalization and selection of fold-changed genes were performed using GeneSpringGX 7.3. The averages of the average of normalized control channel intensity. Functional annotation of genes was performed according to Gene OntologyTM Consortium (http://www.geneontology.org/index.shtml) by GeneSpringGX 7.3. Gene classification was based on searches done by BioCarta (http://www. biocarta.com/), GenMAPP (http://www.genmapp.org/), DAVID (http://david.abcc.ncifcrf.gov/), and Medline databases (http://www.ncbi.nlm.nih.gov/)
Results
Agonistic anti-LTβR antibody disrupted SF of FRC
Members of TNF family cytokines including the membrane heterotrimeric form of LTα1β2 and soluble homotrimeric LTα are important for lymphoid tissue architecture. LTα1β2 signaled through the lymphotoxin-β receptor (LTβ R) expressed in FRC [22]. We used an agonistic anti-LTβR antibody to stimulate FRC. After stimulation, SFs form thick cables and were connected to the nuclear periphery with the processes of these flat and spread cells in a control monolayer (Fig. 1). SFs and FRC showing a striking morphological change with retraction and shrink of their processes were not detected in FRC treated with agonistic anti-LTβR antibody, whereas these morphological changes were rarely observed in control antibody (IgG)-treated FRC (Fig. 1). These findings imply that LTβR in LTα1β2 signaling pathway play a powerful role in the inhibition of SF formation and change of morphology in FRC.
Fig. 1.LTβR stimulation induced the change of FRC stress fiber morphology and cell shape. FRCs were treated with normal IgG and agonistic anti-LTβR antibody (1 μg/ml) for 24 hr, were fixed with 4% formaldehyde in PBS, were washed with PBS and examined for changes in F-actin distribution (phalloidin staining).
LTα signal was partially maintained SF and morphology in FRC
Next, we examined the roles of another member, LTα in the SF formation. Stimulation of FRCs with LTα was unable to induce the appearance of SFs and such cells sometimes revealed bright areas of staining around nucleus (Fig. 2). However, SF was partially maintained in peripheral region of cell and FRC morphology was also maintained (Fig. 2).
Fig. 2.LTα partially disrupted stress fiber. FRCs were treated LTα (10 ng/ml) for 24 hr, fixed, washed and stained for actin stress fiber.
TNFα pathway augmented SF in FRC
The actin cytoskeletal arrangement plays a critical role in maintaining both intracellular and intercellular cell physiological functions. LTα shares its receptors with TNFα (TNFRI and TNFRII) and, thus, the two ligands elicit similar effects [5]. Thus, to examine whether TNFα signaling plays a critical role in SF and morphological alteration in FRC, we treated the cells with TNFα. Upon TNFα stimulation, F-actin is reorganized to form thick SFs across the cell (Fig. 3). Thin SFs are present in non-stimulated FRCs. TNFα induced cell compacted morphology and an increase in SFs that were aligned parallel with the axis of the cells (Fig. 3).
Fig. 3.TNFα (100 ng/ml) augmented stress fiber construction in FRCs. FRCs on chamber slides were stimulated with TNFα for 24 hr, were fixed with 4% formaldehyde in PBS, were washed with PBS and stained for actin stress fiber.
LTβR signal was more critical than LTα signal for the formation of SF in FRC
Fig. 1 depicts that LTβR activation is involved in SF alteration. LT members including LTα, TNFα, and LTα1β2 are involved in SF alteration. However, LTα and TNFα maintained or promoted the formation of SF in FRCs. Thus, to examine whether LTβR signaling plays a critical role in SF formation and morphological alteration in FRC, we treated the cells with LTα + anti-LTβR antibody to anti-LTβR antibody. We were unable to induce the appearance of SFs by incubating the cells with LTα+anti-LTβR antibody to anti-LTβR antibody (Fig. 4). Anti-LTβR antibody treatment significantly reduced these LTα-induced effects on actin cytoskeletal rearrangement. Furthermore, combined stimulation with LTα+anti-LTβR antibody induced cell configuration similar to the effect of anti-LTβR antibody, alone (Fig. 4).
Fig. 4.Combined stimulation using agonistic anti-LTβR antibody (1 μg/ml) and LTα (10 ng/ml) suppresses stress fiber construction in FRCs. FRCs on chamber slides were stimulated with LTα and agonistic anti-LTβR antibody, and stained for actin stress fiber.
LTβR signal was more critical than TNFα signal for the formation of SF in FRC
As shown in fig. 4, the signaling via LRβR followed by the activation of downstream pathways played a powerful role in the suppression of SF and induction of morphological change. To investigate effect of TNFα signaling shared with LTα receptor for SF distribution in FRC, we stimulated TNFα +agonistic anti-LTβR antibody-treated FRC. As expected, upon TNFα+agonistic anti-LTβR antibody stimulation, the appearance of SFs were absent in TNFα+agonistic anti-LTβR antibody-treated FRC (Fig. 5). Moreover, dramatic changes in FRC morphology was shown in TNFα+agonistic anti-LTβ R antibody-treated FRC (Fig. 5). However, these changes ranged from partial retraction and shrink of processes and did not occur in a synchronous manner to the effect of anti-LTβR antibody, alone.
Fig. 5.Combined stimulation using agonistic anti-LTβR antibody (1 μg/ml) and TNFα (100 ng/ml) suppresses stress fiber construction in FRCs. FRCs on chamber slides were stimulated with TNFα and agonistic anti-LTβR antibody, and stained for actin stress fiber.
SF of FRC required MLC phosphorylation
The reorganization of actin cytoskeleton is tightly linked to myosin-driven contraction initiated by MLC phosphorylation [11]. MLC phosphorylation is controlled by myosin light chain kinase (MLCK) [6] and myosin light chain phosphatase (MLCP) [16]. In the next series of experiments, we evaluated FRC SF in the presence of the pharmacological inhibitor shown to prevent MLC phosphorylation. As expected, incubation with the inhibitor of MLCK (ML-7), significantly attenuated actin SF formation in FRC (Fig. 6A). SF formation is facilitated by inducing contractility through regulation of the level phosphorylation of MLC or by the inactivation of the MLCP. We studied the effect of agonistic anti-LTβR antibody on MLC phosphorylation by immuostaining, as the components of SF in FRC. As shown in Figure 6B, agonistic anti-LTβR antibody reduces MLC phosphorylation. Moreover, SF was reduced and cell plasticity was occurred (Fig. 6B). Together, these results demonstrate that the LTβR signaling is linked to mysoin in FRC. Next, we tested anti-LTβR antibody effect on myosin phosphatase target subunit 1 (MYPT1) phosphorylation/inactivation, which is a 130 kDa subunit of a trimeric holoenzyme of MLCP involved in the targeting of MLCP to myosin filaments. As shown in Fig 6C, anti-LTβR antibody reduced MYPT1 phosphorylation and inactive form of MYPT1 was completely blocked. Together, these results demonstrate that the LTβR pathway is involved in MLC phosphorylation in FRC.
Fig. 6.LTβR is linked to myosin on stress fiber formation in FRC. FRCs were treated with ML7 (5 μM) and exhibited a marked decrease in actin stress fibers (A). FRCs on chamber slides were stimulated with agonistic anti-LTβR antibody, and stained for pMLC (B). FRCs were treated with agonistic anti-LTβR antibody (1 μg/ml) for 24 hr and evaluated for change in protein level of pMYPT1 (C). GAPDH was probed as a loading control.
LTβR signal was leaded to change of the expression of genes linked to the formation of SF
To investigate the alteration of gene expression in the FRC on agonistic anti- LTβR antibody, we analyzed via DNA chip assay for transcript changes in agonistic anti-LTβR antibody-treated FRC compared to normal IgG-treated FRC. Our results reveal that agonistic anti-LTβR antibody in FRC (p<0.05) influenced the expression of 887 transcripts regulated minimally by greater than or equal to a 2-fold change to total probe # 39,429. Of the regulated genes, 391 were up-regulated and 496 were down-regulated. Actin bundle held together by α-actinin, fascin, espin, filamin and myosin II bundle composes of SF (14). Of the regulated genes, genes related to myosin and actin component were downregulated. However, there was no change in other factors (Table 1). These suggest that myosin is involved in SF change in agonistic anti-LTβR antibody treated FRC.
Table 1.*Regulation was decided by normalized values (i.e., values equal 2 fold) as Normal regulation and (values below 0.5 fold) as Down regulation. #Normalization was the ratio between anti-LTβR antibody treated sample value vs control value.
Discussion
LN is structured by a complicated 3-dimensional network of stromal cells, connective tissue and extracellular matrix (ECM) fibers. Stromal cell adhesion to the ECM occurs at specialized sites where adhesion receptors bridge between the ECM and the actin cytoskeleton, via a network of scaffold and signaling proteins [3, 7]. LN stromal cells play a role of the scaffold within which the immune cells of the LN can co-localize and dynamically interact [4]. FRCs produce the conduit network ensheathed with the reticular fibers and ECM components, and form a continuous frame work among cells in the T-cell zones of LN [19]. Scaffold structure of cell is important for cell biological and physiological function, maintenance of organ structure. The organization and dynamics of these structures are tightly regulated by associated signaling components, which induce deterioration of SFs [20]. Many of the protein-protein interactions essential for building the interaction scaffold are mediated by the binding of ligand proteins to partner molecules. LTαβ is closely linked to the maintenance of LN network and architecture. In this study, we have illustrated the SF distribution and identified LTβR as a main regulator of SF formation in FRCs. The used concentration of agonistic anti-LTβR antibody (1 ug/ml), LTα (10 ng/ml) and TNF-α (100 ng/ml) was referred to previous report[9]. Fibers look like a cage that surrounds the nucleus in FRC (Fig. 1). Moreover, a very delicate, barely perceptible line staining for actin appears under the plasma membrane in control monolayer. Anti LTβR antibody affected that FRC lose the SFs connecting the processes of the cell (Fig. 1). Severely affected cells assume a dendritic configuration. In the periods after LTα addition, cells showed partial retraction. Such cells sometimes revealed bright globular areas of staining within the cytoplasm, and the periphery of the plasticity cells showed SF (Fig. 2). Although LTα and TNF-α share common receptors (TNFRI and TNFRII), the results of each LTα or TNF-α stimulation were different (Fig. 2, Fig. 3). LTα, a member of the TNF family, is synthesized primarily by activated T. It is expressed in either secreted or membrane-bound form, each exhibiting different affinity for various receptors. The secreted soluble homotrimeric form binds to both TNFR1 and TNFR2 receptors with high affinity, whereas the transmembrane heterotrimeric form (one LTα plus two LTβ) selectively binds the LTβ receptor (LTβR) with high specificity. Recombinant LTα was used in this study. Thus it was thought to be difficulty for the formation of exact homotrimeric LTα form. Thus it seems that signal intensity was too weak to activate cells. In treatments combined with LTα+ anti-LTβR antibody or TNFα+LTβR antibody, dramatic changes of SF and morphology were shown in FRC (Fig. 4, Fig. 5). Rare SFs were present in FRCs. These changes ranged from partial retraction and shrink of processes and occurred in a synchronous manner in agonistic anti LTbR antibody treated FRC. Considerable evidences indicate that myosin bundle is a component of SFs [8. 21, 23]. To address the possibility that the SF alteration in FRC is linked in myosin, FRC was stimulated with ML7 (MLCK inhibitor). As shown in Figure 6A, FRC had become dendritic configuration and FRC devoid of SF was observed. This great plasticity of FRCs is thought to be taken advantage of the supply of the proper character upon sensing immune responses. Moreover, p-MYPT1, inactive form of myosin light chain phosphatse (MLCP), was completely deduced in agonistic anti LTβR antibody treated FRC (Fig. 6B). Collectively, these results revealed that LTβR signaling pathway was involved in myosin mediated cytoskeletal remodeling in FRC.
참고문헌
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