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The Journal of Immunology, 2004, 172: 7459-7465.
Copyright © 2004 by The American Association of Immunologists

Lymphotoxin-{beta} Receptor Activation by Activated T Cells Induces Cytokine Release from Mouse Bone Marrow-Derived Mast Cells1

Peter Stopfer, Daniela N. Männel and Thomas Hehlgans2

Department of Immunology, University of Regensburg, Regensburg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Lymphotoxin-{beta} receptor (LT{beta}R) signaling is known to play a key role in embryonic lymphoid organ formation as well as maintenance of lymphoid architecture. Activation of the LT{beta}R is induced by either the heterotrimeric lymphotoxin-{alpha}1{beta}2 (LT{alpha}1{beta}2) or the homotrimeric LIGHT (homologous to lymphotoxins, exhibits inducible expression, and competes with HSV gpD for herpes virus entry mediator, a receptor expressed by T lymphocyte). Both ligands are expressed on activated lymphocytes. As mast cells reside in close proximity to activated T cells in some inflammatory tissues, we examined the expression of LT{beta}R on bone marrow-derived mast cells and asked whether the LT{beta}R-ligand interaction would allow communication between mast cells and activated T cells. We found that mast cells express LT{beta}R at the mRNA as well as at the protein level. To investigate LT{beta}R-specific mast cell activation, the LT{beta}R on BMMC from either wild-type or LT{beta}R-deficient mice was stimulated with recombinant mouse LIGHT or agonistic mAbs in the presence of ionomycin. LT{beta}R-specific release of the cytokines IL-4, IL-6, TNF, and the chemokines macrophage inflammatory protein 2 and RANTES was detected. Moreover, coculture of mast cells with T cells expressing the LT{beta}R ligands also entailed the release of these cytokines. Interference with a specific LT{beta}R inhibitor resulted in significant suppression of mast cell cytokine release. These data clearly show that LT{beta}R expressed on mast cells can transduce a costimulatory signal in T cell-dependent mast cell activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lymphotoxin-{beta} (LT{beta}) 3 receptor (LT{beta}R) is a member of the TNF receptor superfamily. To date, two functional ligands have been identified that selectively bind to LT{beta}R. It has been shown that the cell surface-bound heterotrimeric LT{alpha}1{beta}2 complex, as well as LIGHT (homologous to lymphotoxins, exhibits inducible expression, and competes with HSV gpD for herpes virus entry mediator (HVEM), a receptor expressed by T lymphocyte), a membrane-anchored homotrimeric complex, are capable of signaling through the LT{beta}R. Both ligands are expressed on activated lymphocytes (1, 2, 3, 4, 5). The LT{beta}R pathway is involved in organization of lymphoid tissues, lymph nodes, and Peyer’s patches during embryogenesis and secondary lymphoid structures in the adult, which can form anywhere in the body at sites of inflammation. Moreover, LT{beta}R is critically involved in the maintenance of spleen architecture, especially the follicular dendritic cell network, and that of lymphoid organs (6, 7). Interestingly, the LT{beta}R is prominently expressed on stromal cells in lymphoid and visceral tissues and on monocytes, but not on lymphocytes (4, 8). This leads to the possibility that the LT{beta}R-LT system may represent a means of communication for activated lymphocytes with their neighboring receptor-positive stroma cells or monocytes/macrophages.

Mast cells are known to be essential effector cells in allergic diseases and contribute to chronic inflammatory processes. They have also been shown to play an important role in host defense against bacterial infections (9, 10). Being mostly located in close proximity to epithelial surfaces, mast cells have a strategic tissue distribution for optimal interaction with the environment (11). Mast cell functions are mediated by a number of preformed molecules, newly synthesized lipid mediators, and several cytokines and chemokines that are secreted upon activation (12). To date, it has been documented that mast cells can undergo degranulation during T cell-mediated inflammatory processes. Moreover, in some morphological studies mast cells were found to reside in close proximity to T cells in inflamed tissues and allergic reactions. This close apposition between mast cells and T cells has led investigators to propose a functional relationship between these two cell populations that might facilitate elicitation of the immune response (13).

Because of their close proximity to mast cells and as activated T cells express both LT{alpha}1{beta}2 and LIGHT, we investigated whether bone marrow-derived mast cells (BMMC) express LT{beta}R and whether the LT{beta}R-LT ligand system could allow communication between activated T cells and mast cells. BMMC were found to express LT{beta}R and released cytokines and chemokines in a LT{beta}R-specific manner upon stimulation with either recombinant mouse (m) LIGHT, agonistic mAbs against the mLT{beta}R, or activated T cells. Thus, the LT{beta}R expressed on BMMC can serve the interaction between activated T cells and mast cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of BMMC

BMMC were derived from C57BL/6 (LT{beta}R+/+ and LT{beta}R–/–) and BALB/c mice. C57BL/6 LT{beta}R–/– mice were provided by K. Pfeffer(Institute of Medical Microbiology, University of Duesseldorf, Duesseldorf, Germany), C57/BL6 wild-type (LT{beta}R+/+) and BALB/c mice were obtained from Charles River Breeding Laboratories (Sulzfeld, Germany). The animals were bred in the animal facility at University of Regensburg and were used at the age of 2–3 mo. Mice were sacrificed by cervical dislocation, femurs were removed, and bone marrow cells were harvested by flushing the femurs with RPMI 1640 medium (Sigma-Aldrich, Steinheim, Germany). The BMMC cultures were established at a density of 3 x 106 cells/ml in IMDM (Life Technologies/Invitrogen, Karlsruhe, Germany), supplemented with 20% FCS (Pan Biotech, Aidenbach, Germany; inactivated for 30 min at 56°C), 100 U/ml penicillin, 100 µg/ml streptomycin, 20 U/ml IL-3, and 200 ng/ml stem cell factor (SCF; c-Kit ligand, provided by L. Hültner (GSF National Research Center for Environment and Health, Munich, Germany)). Nonadherent cells were transferred to fresh medium every 2–3 days for a total of at least 28 days to remove adherent macrophages and fibroblasts.

Flow cytometry

Expression of mLT{beta}R on BMMC was detected by flow cytometry on a FACStarPlus (BD Biosciences, San Jose, CA) using the FITC-coupled specific rat anti-mLT{beta}R mAb 5G11b and a rat IgG2a-FITC (BD Biosciences, Heidelberg, Germany) as isotype control. For flow cytometric analysis of SCF receptor (c-Kit) expression on BMMC anti-CD117-PE-coupled Ab (BD Biosciences) was used. For FACS analysis 1 x 106 BMMC were incubated with 100 µl of a 1 mg/ml solution of mIgG on ice for 30 min to block FcRs, washed twice with PBS (containing 10% heat-inactivated FCS), and incubated with the given Ab (10 µg/ml) for 30 min on ice. Before FACS analysis the BMMC were washed twice with PBS (containing 10% heat-inactivated FCS). For expression studies of mLIGHT and LT{alpha}1{beta}2, activated T cells (1 x 106) were incubated for 30 min on ice with an FITC-coupled LT{beta}R:Ig fusion protein (10 µg/ml). Before FACS analysis the T cells were washed twice with PBS (containing 10% heat-inactivated FCS).

Immunohistochemistry

For toluidine blue staining BMMC were centrifuged onto glass slides in a cytofuge (Shandon, Pittsburgh, PA), incubated for 5 min in a solution containing 1% toluidine in methanol, and washed with distilled water. The cells were then examined using light microscopy. For May-Grünwald-Giemsa staining, cytospins of BMMC were incubated for 4 min in concentrated May-Grünwald solution, washed briefly, kept in Giemsa solution for 6 min, and washed with distilled water before examination using light microscopy.

Abs and recombinant mLIGHT

Recombinant mLIGHT was expressed and purified as previously described for human LIGHT (14). For stimulation experiments the rat anti-mLT{beta}R mAb 5G11b (rat IgG2a) was used (15).

Cell culture

The mouse T cell lines, PMMI (4) and EL4 5D3 (16), were cultured in RPMI 1640 containing 10% heat-inactivated FCS, gentamicin, and 2-ME. BFS-1 (17), a mouse fibrosarcoma cell line, was cultured in RPMI 1640 containing 10% heat-inactivated FCS. L138.8A (18), a mouse mast cell line, was cultured in IMDM (Life Technologies/Invitrogen), 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 20 U/ml IL-3, and 2-ME.

RNA purification and PCR amplification

Total RNA from cultured cells was obtained by using TRIzol reagent (Life Technologies/Invitrogen), according to the manufacturer’s instructions. The RNA was quantified by using Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) according to the manufacturer’s instructions. For the PCR, total RNA (1 µg) from each sample was reverse transcribed in a total volume of 40 µl using an RT system (Promega, Mannheim, Germany) according to the manufacturer’s instructions. The reaction was conducted at 42°C for 15 min and 99°C for 5 min, then cooled to 4°C. The reaction mixture was stored at –20°C until further use.

The amplification was conducted in a 2400 PerkinElmer cycler (PerkinElmer, Wellesly, MA). Each cDNA sample (5 µl) was amplified in 50 µl of a reaction mixture containing 5 µl of PCR buffer (10-fold concentrated; Roche, Mannheim, Germany), 4 µl of deoxynucleotide triphosphate set (PCR grade; 10 mmol; Roche), 1 µl of Taq DNA polymerase (5 U/ml; Roche), 0.2 µl each of 3' and 5' primers (100 µmol/ml; Metabion, Martinsried, Germany), and 34.6 µl of H2O. The sequences of the primers used for PCR amplification were: mLT{beta}R: 5' primer, 5'-GCC GAA GCT TCT GGT GGC CTC TCA GCC CCA G-3'; 3' primer, 5'-GCC GGG ATC CGC TCC TGG CTC TGG GGG ATT-3' (Metabion); mouse herpes virus entry mediator (mHVEM): 5' primer, 5'-GGA GGA TCC GGT GGT TGT GCT GTT GGT CCC AC-3'; 3' primer, 5'-CAG AAG CTT TCT GCC CAG CCC TCA TGC AGA CAG-3' (Metabion); and {beta}-actin: 5' primer, 5'-TGA CGG GGT CAC CCA CAC TGT-3'; 3' primer, 5'-CTA GAA GCA TTT GCG GTG GAC-3' (Metabion). Annealing temperatures for each primer pair were: mLT{beta}R, 60°C; mHVEM, 60°C; and {beta}-actin, 58.5°C. Samples were heated for 5 min at 95°C, and the cycles were 95°C for 45 s, annealing temperature for 45 s, and 72°C for 45 s for 35 cycles. At the end, the reaction mix was kept at 72°C for 7 min. Negative and positive controls were amplified in each PCR experiment. To confirm the use of equal amounts of RNA and to verify a uniform amplification process, {beta}-actin mRNA was amplified in each assay. Aliquots of the samples were analyzed by electrophoresis on a 1.5% agarose gel containing ethidium bromide. The DNA products were visualized by UV fluorescence and photographs of the gels were taken.

In vitro cell stimulation

Stimulations were performed in triplicate using 48-well plates with 106 cells in a final volume of 1 ml of culture medium RPMI 1640 (Sigma-Aldrich) supplemented with 10% FCS (inactivated for 30 min at 56°C), 100 U/ml penicillin, 100 µg/ml streptomycin, including 0.5 µM ionomycin (Sigma-Aldrich), and either recombinant mLIGHT or rat anti-mLT{beta}R mAb 5G11b at the given concentrations. As a negative/positive control, BMMC were treated with either rat IgG2a (10 µg/ml) or PMA plus ionomycin (2 µM). After 24 h cytokines in the supernatants were tested by ELISA.

Coculture of activated T cells and BMMC

T cells were stimulated by preincubation with PMA (Sigma-Aldrich) and ionomycin in the stated concentrations for 14 h, washed (three times with RPMI 1640), and cocultured in increasing ratios of T cells to BMMC for 24 h in RPMI 1640 (Sigma-Aldrich) supplemented with 10% FCS (inactivated for 30 min at 56°C), 100 U/ml penicillin, and 100 µg/ml streptomycin. Cytokines were tested in the supernatants by ELISA.

Measurement of {beta}-hexosaminidase release

BMMC (1 x 105 cells/100 µl/well) were suspended in Tyrode’s buffer (TB; Sigma-Aldrich; 37°C) supplemented with 0.05% gelatin (modified TB). Release modulators (mLIGHT, 5G11b, or PMA-activated T cells) were added and incubated for 10 min at 37°C, followed by 0.5 µM ionomycin, dissolved in 50 µl of modified TB as release inducer (except for the coculture of activated T cells and BMMC, to which no additional ionomycin was added), and incubated for an additional 30 min at 37°C. After centrifugation (1200 rpm, 4°C), supernatants were harvested, and the cell pellet was lysed with 200 µl/well 0.5% Triton X-100 (in distilled water). Aliquots (20 µl) of supernatants or cell lysates were incubated with 50 µl of 1 mM p-nitrophenyl-N-acetyl-{beta}-D-glucosamine (enzyme substrate) dissolved in 0.1 M sodium citrate (pH 4.5) for 60 min at 37°C. Then 250 µl of a 0.1 M Na2CO3/0.1 M NaHCO3 buffer (pH 10) was added, and absorbance was measured at 410 nm in a precision microplate ELISA reader (MWG Biotech, Ebersberg, Germany). Values were expressed as the percentage of total {beta}-hexosaminidase (total {beta}-hexosaminidase = individual supernatant + corresponding lysate).

Cytokine ELISA

Mouse IL-4, IL-6, TNF, macrophage inflammatory protein 2 (MIP-2), and RANTES levels were measured with ELISA kits (R&D Duo Sets; R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol.

LT{beta}R:Ig fusion protein expression

The expression and purification of the fusion protein LT{beta}R-Ig composed of the extracellular domain of mouse LT{beta}R fused to the Fc domain of human IgG1 have been described recently (17).

Statistical analysis

Statistical analysis was performed using Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
BMMC express mLT{beta}R

BMMC were generated by culturing bone marrow cells from either C57BL/6 wild-type (LT{beta}R+/+) or LT{beta}R–/– mice in the presence of IL-3 and SCF. After 4 wk of culture the cells demonstrated characteristic toluidine blue and May-Grünwald staining of mast cells granules (>96%; data not shown). Also, the cells derived from both LT{beta}R+/+ and LT{beta}R–/– mice showed high levels of c-Kit as determined by flow cytometric analysis for CD117 (data not shown). LT{beta}R+/+ BMMC showed a higher expression of c-Kit at this time of culture compared with LT{beta}R–/– BMMC. After another 2 wk of culture, the BMMC derived from both LT{beta}R+/+ and LT{beta}R–/– mice showed identical high levels of c-Kit (>98%) as determined by flow cytometric analysis of CD117 (Fig. 1A), indicating a pure BMMC population. Mast cells are the only cells of hemopoietic origin that are c-Kit positive as mature cells (19). Also, the cells demonstrated characteristic May-Grünwald (Fig. 1B) and toluidine blue (data not shown) staining of mast cell granules (>96%). Thus, both LT{beta}R–/– and LT{beta}R+/+ stem cells are capable of developing into BMMC in the presence of IL-3 and SCF within 6 wk.



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  		FIGURE 1. Generation of LT{beta}R+/+ and LT{beta}R–/– BMMC. A, Flow cytometric analysis of BMMC for surface expression of CD117. BMMC from LT{beta}R+/+ or LT{beta}R–/– mice were stained with anti-CD117-PE mAb (solid line, LT{beta}R+/+; dotted line, LT{beta}R–/–) or PE-coupled isotype control mAb (shaded area). B, May-Grünwald staining of BMMC. Upper panel, LT{beta}R+/+ BMMC; lower panel, LT{beta}R–/– BMMC. Representative data from one of three independent experiments are shown. BMMC were generated from LT{beta}R+/+ and LT{beta}R–/– mice in three independent experiments.

 
To investigate whether these cells express LT{beta}R on their surface, LT{beta}R–/– and LT{beta}R+/+ BMMC were tested by FACS analysis using the specific rat anti-mLT{beta}R mAb 5G11b (Fig. 2A). Flow cytometric analysis clearly showed that LT{beta}R–/– BMMC were devoid of LT{beta}R protein on the cell surface, with no difference in background fluorescence intensity between the isotype-matched control Ab (rat IgG2a) and the 5G11b. LT{beta}R+/+ BMMC clearly expressed LT{beta}R, as demonstrated by the fluorescence shift between the isotype control and 5G11b staining. Similar results were obtained with BMMC from BALB/c mice (data not shown). FACS analysis for LT{beta}R expression was also performed with the mouse mast cell line L138.8A, but these cells were found to be negative (data not shown). To confirm this result, RT-PCR analysis for LT{beta}R was performed using LT{beta}R+/+ BMMC (Fig. 2B). As a negative control cDNA from the mouse T cell line PMMI and as a positive control cDNA from mouse fibrosarcoma BFS1 cells were used. RT-PCR analysis supported the staining results by confirming LT{beta}R mRNA expression in BMMC derived from LT{beta}R+/+ mice. Again, no mRNA for LT{beta}R was detected in the mast cell line L138.8A. To test whether cytokine release of stimulated BMMC was LT{beta}R specific, we checked ionomycin-activated (0.5 µM) LT{beta}R+/+ BMMC for HVEM expression by RT-PCR. As shown in Fig. 2C, these stimulated BMMC failed to express HVEM. As a positive control cDNA from the mouse T cell line PMMI and as a negative control cDNA from mouse fibrosarcoma BFS1 cells were used.



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  		FIGURE 2. Expression of LT{beta}R on BMMC. A, Flow cytometric analysis of BMMC for surface LT{beta}R expression. BMMC from LT{beta}R+/+ or LT{beta}R–/– mice were stained with 5G11b-FITC (solid line, LT{beta}R+/+; dotted line, LT{beta}R–/–) or rat IgG2a-FITC as isotype control (shaded area). B, RT-PCR analysis of the mouse mast cell line L138.8A, mouse fibrosarcomas BFS1 (positive control), the mouse T cell line PMMI (negative control), and BMMC from LT{beta}R+/+ for LT{beta}R expression. C, RT-PCR analysis of ionomycin-stimulated (0.5 µM) BMMC, the mouse T cell line PMMI (positive control), and the mouse fibrosarcoma cell line BFS1 (negative control) for HVEM expression. Representative data from one of three independent experiments are shown.

 
These data clearly demonstrate that BMMC express LT{beta}R at both mRNA and protein levels and fail to express HVEM at the mRNA level. To date, LT{beta}R has only been known to be expressed on stromal cells, in lymphoid and visceral tissues (e.g., epithelial cells), and on monocytes (3, 4, 5, 8, 20).

Mouse LIGHT and agonistic Abs to the LT{beta}R are costimulators for cytokine release, but not for degranulation of BMMC

We further investigated whether selective LT{beta}R stimulation activates BMMC for cytokine secretion, because it has been reported that mast cell activation can lead to the secretion of preformed as well as newly synthesized cytokines and chemokines (12). BMMC from LT{beta}R+/+ and LT{beta}R–/– mice were activated following a standard protocol using ionomycin (0.5 µM, 24 h) in the absence or the presence of agonistic rat anti-mLT{beta}R Abs 5G11b. The supernatants were tested for different cytokines/chemokines (IL-4, IL-6, TNF, MIP-2, and RANTES) using specific ELISAs. Fig. 3A shows that there was no difference in the general activation state between BMMC from LT{beta}R–/– and LT{beta}R+/+ mice, because positive control stimulation (2 µM PMA plus ionomycin) induced similar cytokine levels in both cell populations. Ionomycin alone was also able to induce low, but significant, release of these cytokines/chemokines from both BMMC populations



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  		FIGURE 3. Costimulatory effect of agonistic anti-LT{beta}R Abs or LIGHT on cytokine release from BMMC. BMMC derived from either LT{beta}R+/+ ({blacksquare}) or LT{beta}R–/– ({square}) mice were stimulated with ionomycin (0.5 µM) and the stated amounts of agonistic anti-LT{beta}R Abs 5G11b (A) or recombinant LIGHT (B). As a positive control, 2 µM ionomycin plus PMA was used; as a negative control, culture medium was used. Mean values of triplicate determinations ± SD of cytokine concentrations (IL-6, TNF, IL-4, MIP-2, and RANTES) were measured in the supernatants using specific ELISAs. Representative data from one of four independent experiments are shown. Statistical analysis was performed using Student’s t test. *, p < 0.05.

 
However, costimulation with LT{beta}R-specific agonistic Abs 5G11b induced significantly higher extracellular cytokine/chemokine levels from LT{beta}R+/+ than from LT{beta}R–/– BMMC, demonstrating the LT{beta}R specificity of the cytokine/chemokine release. The cytokine/chemokine release was found to be dependent on the 5G11b dose. Although IL-4, IL-6, TNF, and RANTES were detected in high concentrations compared with the positive PMA/ionomycin control, MIP-2 was found in relatively small amounts in the supernatants (up to 100–115 pg/ml after stimulation with 20 or 50 µg of 5G11b/ml). In the absence of the calcium ionophore ionomycin, 5G11b induced no detectable cytokine release (data not shown), indicating the requirement for a calcium-dependent costimulation for LT{beta}R-induced cytokine/chemokine release from BMMC. When BMMC were treated with recombinant mLIGHT instead of the LT{beta}R-specific agonistic Abs 5G11b, similar results were obtained (Fig. 3B). Again, in the absence of ionomycin, mLIGHT did not induce any detectable cytokine release from BMMC (data not shown). Additionally, we used rat IgG2a control Abs in the concentrations of 1, 10, and 25 µg/ml together with ionomycin-stimulated (0.5 µM) LT{beta}R+/+ or LT{beta}R–/– BMMC. Cytokine release in such control cultures was in the same range as in cultures of LT{beta}R–/– BMMC stimulated with 5G11b/mLIGHT (Fig. 3; data not shown), indicating that measured cytokine release after 5G11b treatment was not due to Fc{gamma}R stimulation.

To test whether stimulation of the LT{beta}R on BMMC in the presence of ionomycin is capable of inducing mast cell degranulation, secretion of {beta}-hexosaminidase, as a marker for the short term release of preformed mediators such as histamine, was measured. BMMC from LT{beta}R+/+ and LT{beta}R–/– mice were treated with increasing amounts of either 5G11b or mLIGHT in the presence of ionomycin. Neither stimulation with 5G11b (Fig. 4) nor that with mLIGHT (data not shown) had any significant effect on BMMC degranulation. However, PMA/ionomycin treatment, used as a positive control, induced significant release of {beta}-hexosaminidase (Fig. 4).



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  		FIGURE 4. {beta}-Hexosaminidase release of BMMC after stimulation with agonistic anti-LT{beta}R Abs. BMMC derived from either LT{beta}R+/+ ({blacksquare}) or LT{beta}R–/– ({square}) mice were stimulated with ionomycin (0.5 µM) and the stated amounts of agonistic anti-LT{beta}R Abs 5G11b. As a readout of degranulation, {beta}-hexosaminidase release in the supernatants was measured. As a positive control, 2 µM ionomycin plus PMA were used; as negative control, culture medium was used. Mean values of triplicate determinations ± SD of {beta}-hexosaminidase concentrations are given. Representative data from one of three independent experiments are shown.

 
Cocultivation of stimulated T cells with BMMC leads to cytokine release from BMMC

We investigated whether LT{alpha}1{beta}2 and/or LIGHT on the surface of activated T cells are capable of inducing mast cell cytokine release by activating the LT{beta}R on mast cells. Such a mechanism would require cell-to-cell contact. Cells of the EL4 5D3 mouse T cell line were activated for 14 h in the presence of PMA plus ionomycin to induce LT{alpha}1{beta}2 and LIGHT expression (5). Such activated EL4 5D3 cells stained positively (48%) for LT{beta}R:Ig-FITC binding, as shown by flow cytometric analysis (Fig. 5A). PMA and ionomycin was removed by extensive washing before adding the activated EL4 5D3 cells to the BMMC cultures. After cocultivation of different ratios of activated EL4 5D3 cells with BMMC from either LT{beta}R+/+ or LT{beta}R–/– mice for 24 h, the supernatants were analyzed for cytokines. Activated EL4 5D3 cells were also cultured separately, and the baseline cytokine level from these supernatants was subtracted from the cytokine levels from cocultures. As shown in Fig. 5B, activated EL4 5D3 cells were equally able to induce the release of IL-4, IL-6, TNF, MIP-2, and RANTES from LT{beta}R+/+ BMMC as the soluble costimulators 5G11b and LIGHT. IL-6, IL-4, and MIP-2 reached comparable levels upon stimulation with activated T cells as with 5G11b or LIGHT, whereas the levels of RANTES and especially TNF were much lower. BMMC from LT{beta}R–/– mice also secreted a certain amount of cytokines, indicating that the LT{beta}R pathway is not the only way in which activated T cells stimulate mast cells. However, the difference in cytokine release from LT{beta}R+/+ and LT{beta}R–/– BMMC after coculture with activated T cells was clear and amounted to ~30–40%.



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  		FIGURE 5. Cocultivation of activated EL4 5D3 with BMMC leads to LT{beta}R-dependent cytokine production. A, Flow cytometric analysis of EL4 5D3, activated with PMA (500 ng/ml) plus ionomycin, for the expression of LT{alpha}1{beta}2 and/or LIGHT was performed by staining with an FITC-coupled LT{beta}R:Ig fusion protein. LT{beta}R:Ig stained unstimulated EL4 5D3 (shaded area) and PMA-activated EL4 5D3 (solid line). B, Effect of PMA/ionomycin-activated EL4 5D3 cells on cytokine release (IL-6, TNF, IL-4, MIP-2, and RANTES) from BMMC of LT{beta}R+/+ and LT{beta}R–/– mice. Activated EL4 5D3 were added to either LT{beta}R+/+ BMMC ({blacksquare}) or LT{beta}R–/– BMMC ({square}). Cytokine concentrations in the supernatants were measured by ELISA, and the mean ± SD (n = 3) are given. Representative data from one of three independent experiments are shown. Statistical analysis was performed using Student’s t test. *, p < 0.05.

 
To test whether coculture of activated T cells with BMMC also leads to degranulation of mast cells as has been suggested previously (13), we measured {beta}-hexosaminidase release after 30 min of coculture of LT{alpha}1{beta}2- and/or LIGHT-expressing T cells with BMMC from both LT{beta}R+/+ and LT{beta}R–/– mice. In contrast to the previous findings (13), no {beta}-hexosaminidase was released from BMMC from either LT{beta}R+/+ or LT{beta}R–/– mice (Fig. 6) under our conditions, indicating that signaling via the LT{beta}R does not result in degranulation of BMMC.



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  		FIGURE 6. Cocultivation of activated EL4 5D3 with BMMC does not lead to LT{beta}R-dependent degranulation of BMMC. The release {beta}-hexosaminidase from BMMC from LT{beta}R+/+ ({blacksquare}) and LT{beta}R–/– ({square}) mice is shown after coculture with PMA/ionomycin-activated EL4 5D3. As a positive control, 2 µM ionomycin and PMA were used. Mean values of triplicate determinations ± SD of {beta}-hexosaminidase concentrations are given. Representative data from one of three independent experiments are shown.

 
Blocking of LT{beta}R ligands on activated EL4 5D3 inhibited cytokine release from BMMC

To further support the LT{beta}R specificity of mast cell activation, PMA- plus ionomycin-stimulated EL4 5D3 cells were incubated with a functional LT{beta}R inhibitor (LT{beta}R:Ig) to selectively block all ligands of the LT{beta}R on the surface of activated T cells. After extensive washing, the activated and LT{beta}R:Ig-incubated EL4 5D3 cells were cocultured with LT{beta}R+/+ BMMC, and the supernatants were again tested for cytokines. Preincubation of activated EL4 5D3 cells with the LT{beta}R inhibitor before coculture with LT{beta}R+/+ BMMC reduced cytokine levels to the levels released from cocultures of activated EL4 5D3 cells with LT{beta}R–/– BMMC (Fig. 7).



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  		FIGURE 7. Cocultivation of activated EL4 5D3, incubated with LT{beta}R:Ig, with BMMC leads to decreased cytokine production from BMMC. Activated EL4 5D3 T cells were added to either LT{beta}R+/+ BMMC ({blacksquare}) or LT{beta}R–/– BMMC ({square}), or activated EL4 5D3 T cells were preincubated with LT{beta}R:Ig and then added to LT{beta}R+/+ BMMC (). Cytokine concentrations (IL-6, TNF, IL-4, MIP-2, and RANTES) in the supernatants were measured by ELISA, and the mean values of triplicate determinations ± SD are shown. Representative data from one of two independent experiments are shown. Statistical analysis was performed using the Student t test. *, p < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study clearly shows that LT{beta}R is expressed on BMMC, whereas they fail to express HVEM. As LT{beta}R mRNA has been found to be constitutively expressed in mouse tissues from lung, liver, kidney, heart, spleen, and lymph nodes, it was detected in neither T nor B lymphocytes (4, 20). These results were extended by a recent report that mLT{beta}R RNA is also found in mouse tissues of skin, muscle, bone, stomach, small and large intestines, thymus, ovaries, and uterus (21). During the embryonic stage the epithelial cells were shown to be LT{beta}R positive, especially those derived from small and large intestines. LT{beta}R expression was also found on stromal cells of the developing human thymus and on reticular dendritic cells of the thymus and spleen, particularly in the white pulp (22). Also, LT{beta}R expression was demonstrated on follicular dendritic cells (23, 24). Finally, LT{beta}R expression was shown on a cell line of hemopoietic origin, i.e., RAW cells, and on peritoneal mouse macrophages (5). This pattern of LT{beta}R expression indicates that LT{beta}R is predominantly found on cells of nonhemopoietic origin. Therefore, besides monocytes/macrophages, the mast cell is another cell type of hemopoietic origin expressing the LT{beta}R.

The BMMC used in this investigation were derived from C57BL/6 or BALB/c mice by culture in IL-3 and Kit ligand; therefore, they may resemble more closely connective tissue mast cells than BMMC generated in IL-3 alone. In contrast, the L138.8A mast cell line, cultured in the presence of IL-3, represents a very immature mast cell precursor cell type that still has the capability to differentiate toward both mucosal mast cells and connective tissue mast cells (18, 25, 26). This may be the reason for the lack of LT{beta}R expression on L138.8A cells.

Activation of the LT{beta}R on BMMC with either agonistic Abs or recombinant mLIGHT in the presence of ionomycin induced the release of IL-4, IL-6, TNF, MIP-2, and RANTES. A comparable LT{beta}R-specific cytokine release was induced when LT{alpha}1{beta}2- and/or LIGHT-expressing T cells were cocultured with BMMC. As HVEM is not expressed in ionomycin-stimulated BMMC, the measured cytokine levels are specific for LT{beta}R activation. Activation of mast cells by coculture with stimulated T cells has been described previously, but it was not clear which mediators were involved (27, 28). Our results clearly show that the interaction of LT{alpha}1{beta}2 and/or LIGHT exposed on activated T cells with LT{beta}R expressed on mast cells provides a possible means of interaction for these two cell types. B cells may communicate with mast cells in the same way, because B cells are also known to express LT{alpha}1{beta}2 and/or LIGHT (2).

A requirement for additional costimulatory proteins on the surface of activated T cells for LT{beta}R-specific cytokine/chemokine release from BMMC also became clear in our study. This adds to the results of Fig. 3 showing that additional signaling (in this study, calcium signaling) is involved in LT{beta}R-specific cytokine/chemokine release of BMMC. When EL4 5D3 cells were stimulated with a low amount of PMA (100 ng/ml) plus ionomycin, they expressed LT{alpha}1{beta}2 and/or LIGHT to a similar degree as EL4 5D3 cells stimulated with a 5-fold higher amount of PMA plus ionomycin. Although such a relatively weak stimulation still induced IL-6, IL-4, and RANTES release, it failed to induce TNF and MIP-2 release (data not shown). Thus, additional molecules on the cell surface of activated T cells, different from LT{alpha}1{beta}2 and LIGHT, are required for proper T cell-mast cell interaction and seem to be essential for TNF and MIP-2 secretion in this coculture system. No additional ionomycin was needed in the T cell-BMMC cocultures for cytokine release from BMMC. Activation of LT{beta}R on BMMC is certainly not the only means of communication between activated T cells and mast cells, because even LT{beta}R–/– BMMC are capable of releasing a certain amount of cytokines/chemokines after coculture with stimulated T cells. LT{beta}R-specific secretion of IL-6 and IL-4 from BMMC could also be induced with other activated T cells, i.e., PMMI and ESB (data not shown). In general, connective tissue mast cells result from treatment with IL-3, IL-4, and SCF and demonstrate a different cytokine profile than BMMC generated under the influence of IL-3, IL-9, and IL-10 (29). Therefore, BMMC generated under different culture conditions might release different cytokines after LT{beta}R activation.

The cytokines tested in our experiments do not allow classification as typical Th2 or Th1 cytokines. IL-4, prominently known as a Th2 cytokine profile marker affecting a broad range of different cell types, including mast cells, T and B cells, macrophages, and endothelial cells, could cause promotion of survival and growth of BMMC or differentiation of T cells to Th2 cells (30). Because Th2 cells play an important role in allergic reactions, it is possible that LT{beta}R activation on BMMC may play a decisive role in allergic reactions (31, 32). Moreover, mast cells are a critical source of IL-4, which together with IL-6 and TNF, plays a pivotal role in initiating and maintaining the inflammatory response during inflammation, e.g., asthma (33).

The release of IL-6 from BMMC after LT{beta}R stimulation is of interest because it is known that IL-6, in the presence of IL-4, promotes differentiation of T cells toward Th2 cells and simultaneously inhibits polarization to Th1 cells through two independent molecular mechanisms (34). Furthermore, IL-6 is known to be involved in a wide range of immune reactions, e.g., allergic inflammation, host defense against parasites, the synthesis of acute phase response proteins, and growth of hemopoietic stem cells (33, 35). Increased levels of IL-6 have been observed in several diseases, including rheumatoid arthritis, psoriasis, and inflammatory bowel disease. IL-6 is critically involved in experimentally induced autoimmune disease, such as Ag-induced arthritis and experimental allergic encephalomyelitis. All clinical data and the animal models suggest that IL-6 plays a critical role in the pathogenesis of autoimmune diseases (36). The pathogenesis of these diseases is prominently driven by activated T cells, as demonstrated in ulcerative colitis, where T cells highly express LT{beta} on their surface (37). Moreover, LT{beta}R activation is known to be critically involved in the pathogenesis of models of experimental inflammatory bowel disease, collagen-induced arthritis, and experimental autoimmune encephalomyelitis (38, 39, 40, 41, 42). As mast cells are known to be involved in the perpetuation of these diseases, especially rheumatoid arthritis (43), blocking LT{beta}R activation by LT{beta}R:Ig treatment may be beneficial because of the reduced production of IL-6 and other cytokines/chemokines, such as TNF, IL-4, RANTES, and MIP-2, by mast cells. TNF, IL-4, and chemokines are well known to influence the pathogenesis of autoimmune diseases (43).

TNF and MIP-2, even though only released in low levels in our experimental model, may play an important role by controlling the recruitment of neutrophils during T cell-mediated, delayed-type hypersensitivity reaction (44). RANTES, which has been reported to be released upon LT{beta}R activation from A375 melanoma cells (45), acts in a proinflammatory way and could contribute to the chronicity of inflammatory diseases, e.g., ulcerative colitis, at sites where mast cell are enriched (46).

Taken together these data show that the interaction of LT{alpha}1{beta}2 and/or LIGHT on activated T cells and LT{beta}R expressed on mast cells could allow the communication of these two cell types. It is conceivable that LT{beta}R signaling may serve as a coactivating stimulus for mast cells in the course of T cell- or B cell-dependent inflammatory reactions. Mast cells may thus augment inflammatory cascades by the enhanced secretion of cytokines and chemokines with paracrine and/or autocrine functions. Cytokines/chemokines released from mast cells after LT{beta}R activation have been shown to be involved in the pathogenesis of multiple autoimmune and chronic inflammatory diseases, in which critical roles for mast cells as well as for LT{beta}R activation have been documented (35, 36, 38, 43, 46).


   Acknowledgments
 
We thank L. Hültner (GSF National Research Center for Environment and Health, Munich, Germany) for providing SCF and IL-3 and for helpful discussions. We also thank K. Pfeffer (Institute of Medical Microbiology, University of Duesseldorf, Duesseldorf, Germany) for providing the LT{beta}R–/– mice.


   Footnotes
 
1 This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 585-02 TPB2. Back

2 Address correspondence and reprint requests to Dr. Thomas Hehlgans, Department of Immunology, University of Regensburg, Franz Josef Strauss Allee 11, 93042 Regensburg, Germany. E-mail address: thomas.hehlgans{at}klinik.uni-regensburg.de Back

3 Abbreviations used in this paper: LT{beta}, lymphotoxin-{beta}; BMMC, bone marrow-derived mast cell; HVEM, herpes virus entry mediator; LIGHT, homologous to lymphotoxins, exhibits inducible expression, and competes with HSV gpD for HVEM, a receptor expressed by T lymphocytes; LT{beta}R, LT{beta} receptor; m, mouse; MIP-2, macrophage inflammatory protein 2; SCF, stem cell factor; TB, Tyrode’s buffer. Back

Received for publication August 26, 2003. Accepted for publication April 6, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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