Correspondence to: Yang-Xin Fu, Dept. of Pathology, MC6027, The University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637. Tel:773-702-0929 Fax:773-702-6260 E-mail:yfu{at}midway.uchicago.edu.
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Abstract |
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Although several cytokines, including tumor necrosis factor (TNF), can promote the growth of dendritic cells (DCs) in vitro, the cytokines that naturally regulate DC development and function in vivo have not been well defined. Here, we report that membrane lymphotoxin (LT), instead of TNF, regulates the migration of DCs in the spleen. LT-/- mice, lacking membrane LT
/ß and LT
3, show markedly reduced numbers of DCs in the spleen. Unlike wild-type mice and TNF-/- mice that have densely clustered DCs in the T cell zone and around the marginal zone, splenic DCs in LT
-/- mice are randomly distributed. The reduced number of DCs in lymphoid tissues of LT
-/- mice is associated with an increased number of DCs in nonlymphoid tissues. The number of splenic DCs in LT
-/- mice is restored when additional LT-expressing cells are provided. Blocking membrane LT
/ß in wild-type mice markedly diminishes the accumulation of DCs in lymphoid tissues. These data suggest that membrane LT is an essential ligand for the presence of DCs in the spleen. Mice deficient in TNF receptor, which is the receptor for both soluble LT
3 and TNF-
3 trimers, have normal numbers of DCs. However, LTßR-/- mice show reduced numbers of DCs, similar to the mice lacking membrane LT
/ß. Taken together, these results support the notion that the signaling via LTßR by membrane LT
/ß is required for the presence of DCs in lymphoid tissues.
Key Words: membrane lymphotoxin, tumor necrosis factor, dendritic cells, lymphotoxin receptor, migration
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Introduction |
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Soluble lymphotoxin (LT)1 and TNF-
are structurally related homotrimers (LT
3 and TNF-
3) that show similar biological activities by binding to either of the two defined TNF receptors, TNFR-I and TNFR-II, leading to activation of a wide variety of inflammatory and immune responses (1) (2). LT
also exists as a membrane ligand by binding to LTß to form a membrane LT
1ß2 heterotrimer (membrane LT), which shows a high-affinity interaction with LTß receptor (LTßR) but only very low affinity for TNFR-I or TNFR-II. The expression of membrane LT
1ß2 is detected on activated T, B, and NK cells, whereas its receptor is expressed exclusively in nonlymphoid tissues (1) (3) (4) (5). The role of membrane LT and LTßR has been recently revealed by gene targeting. LT
-/-, LTß-/-, and LTßR-/- mice all manifest profoundly defective LN and Peyer's patch development and altered splenic structure and B cell follicles (6) (7) (8) (9). Blocking membrane LT function during mouse ontogeny by injection of a soluble LTßRIg fusion protein or an anti-LTß mAb to pregnant wild-type (wt) mice resulted in the absence of peripheral lymphoid organogenesis in their progeny. Conversely, activation of LTßR with an agonistic mAb could restore LN formation in the LT
-/- mice (10) (11). The data prove that signaling via LTßR by membrane LT on nonlymphocytes is required for lymphoorganogenesis and the formation of the lymphoid tissue microenvironment.
The formation of microenvironment, such as B cell follicles and T/B cell segregation in lymphoid tissue, may depend on the expression of membrane LT on B cells (12) (13) (14) (15). LT may also regulate the localization of various lymphoid and nonlymphoid cells by regulating a series of chemokines in the lymphoid organs. For example, some chemokines produced by stromal cells in B cell follicles direct the polarization of the B cell follicles (16). Although the cell types producing chemokines induced by LT in lymphoid tissue have not been identified, the expression pattern of chemokines in lymphoid tissues resembles the distribution pattern of follicular dendritic cells (FDCs) in B cell follicles and lymphoid dendritic cells (DCs) in T cell zones (2) (16). Although the role of membrane LT in the regulation of B cellrelated events and the maintenance of FDCs is well defined, participation of this regulatory system in DC/T cell events remains unclear. Interestingly, inhibition of the membrane LT pathway has profound effects on several T cellbased disease models, e.g., colitis (17), collagen-induced arthritis (Browning, J.L., and R.A. Fava, unpublished observations), and induction of experimental autoimmune encephalitis (Browning, J.L., and C.L. Nickerson-Nutter, unpublished observation). T lymphocytes are important mediators of immunity, but their function is tightly regulated by DCs (18) (19). One explanation for these observations would be parallel regulation of DC/T cells, similar to that of FDC/B cells, in an LT-dependent fashion (12) (13) (14) (15) (16).
Cytokines, such as GM-CSF and TNF, promote the growth of DCs in vitro, but less is known about the regulation of DC distribution and development in vivo (20) (21). Injection of a pharmacological dose of polyethylene glycolmodified GM-CSF into mice only expands the myeloid-related DC subset (22). Interestingly, GM-CSF-/- or GM-CSFR-/- mice show no significant impairment in the development of splenic DCs, suggesting that this cytokine is not absolutely required for DC development (23). Here, we report that LT-/- or LTßR-/- mice show markedly reduced numbers of splenic DCs but increased numbers of DCs in nonlymphoid tissues. DCs are present in normal numbers and distribution in TNF-/- and TNFR-/- mice. Reconstitution of LT
-/- mice with LT-expressing cells restores the number of DCs in the spleen. On the other hand, removal of LT-expressing cells or blocking membrane LT in wt mice created an impaired DC migration phenotype similar to that seen in LT
-/- mice. These findings strongly suggest that signaling via LTßR by membrane LT is critical for the migration of DCs into lymphoid tissues.
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Materials and Methods |
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Animals.
LT-/- mice (backcrossed to C57BL/6 mice for seven generations) and their wt littermates on a C57BL/6 background were bred under specific pathogen-free conditions as described (6). LTßR-/- mice were provided by Dr. Klaus Pfeffer (Technical University of Munich, Germany) (8). TCR-/-, BCR-/-, RAG-1-/-, TNFR-I-/-, and TNF-/- mice as well as CD3
-transgenic mice were purchased from The Jackson Laboratory. B6-Ly5.1 mice were purchased from Frederick Cancer Center, National Cancer Institute, Bethesda, Maryland. Animal care and use were in accordance with institutional guidelines.
Cell Preparation and Staining.
Splenic DCs were treated and collected basically according to the method developed by Inaba et al. (24). In brief, spleen fragments were digested with 2 mg/ml of collagenase and 100 µg/ml DNase for 30 min at 37°C and then gently pipetted in the presence of 0.01 M EDTA for 1 min. Single-cell suspensions were stained and analyzed by two-color flow cytometry on a FACScanTM (Becton Dickinson). Biotinylated anti-CD11c and CD11b (Mac-1), FITC-conjugated antiI-Ab, anti-CD11c, and anti-CD8 antibody were all obtained from PharMingen.
Immunohistology.
Spleens were harvested, embedded in OCT compound (Miles-Yeda, Inc.), and frozen at -70°C. Frozen sections (610 µm thick) were fixed in cold acetone. Endogenous peroxidase was quenched with 0.2% H2O2 in methanol. After washing in PBS, the sections were stained by first incubating with FITC-conjugated anti-B220 for B cells and biotinylated anti-CD11c for DCs (PharMingen) at 1:50100 dilution. Horseradish peroxidaseconjugated rabbit antiFITC (DAKO Corp.) and alkaline phosphataseconjugated streptavidin (Vector Labs., Inc.) were added 1 h later. Color development for alkaline phosphatase and horseradish peroxidase was performed with an alkaline phosphatase reaction kit (Vector Labs., Inc.) and with 3,3'-diaminobenzidine (Sigma Chemical Co.).
Generation of Reagents that Block Membrane LT Activity.
Anti-LTß antibody and some aspects of the control LTßRIg fusion protein used in this study have been previously described (4). The method for the generation of LTßRIg fusion protein was used as previously described with a minor modification (4). In brief, cDNA encoding the extracellular domain of murine LTßR was isolated by RT-PCR using the sense primer (5'-AAAGGCCGCCATGGGCCT-3') and the antisense primer (5'-TTAAGCTTCAGTAGCATTGCTCCTGGCT-3') from mouse lung mRNA, digested by NcoI/HindIII, and then fused to an IL-3 leader sequence in p30242 vector. The fusion fragment was then subcloned into pX58 vector containing the IE-175 promoter and the Fc portion of human IgG1, which was then transfected into BHK/VP16 cells. The mouse LTßRhuman Ig in culture supernatants was purified on a protein A column. No difference can be found between LTßRhuman Ig in this preparation and a previous LTßRIg preparation in Chinese hamster ovary cells (4). To block membrane LT activity in mice, the LTßRIg or anti-LTß antibody (50100 µg/injection) was given intraperitoneally, and the number of DCs was determined 1014 d later by either flow cytometry or immunohistology.
Cell Transfer.
Bone marrow (BM) cell or splenocyte transfer was performed as previously described (12). In brief, BM-derived DCs (BMDCs) from Ly5.1 mice were obtained by culturing BM cells with GM-CSF (5 ng/ml) and IL-4 (2 ng/ml) according to the procedure developed by Inaba et al. (25). BMDCs (5 x 106) or splenocytes (5 x 107) were intravenously transferred into sublethally irradiated recipient mice (600 rads). Spleens and LN cells were collected for analysis within 24 h after transfer.
Mixed Lymphocyte Reaction.
As stimulating cells, splenocytes from wt or LT-/- mice were isolated by gentle pressure through a cell strainer (Becton Dickinson), or spleen fragments were treated with collagenase as described earlier (24). The stimulating cells were irradiated at 2,000 rads. The LN cells from BALB/c mice were collected by gentle pressure using a cell strainer and cultured in a petri dish for 2 h. The nonadherent LN cells were then harvested and used as the source of responding cells. The different amounts of stimulating cells as indicated and 4 x 105 responding cells were cocultured for 72 h, and [3H]TdR at 1 µCi/ml was added during the last 18 h.
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Results and Discussion |
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Markedly Reduced Numbers of DCs in LT-/- Mice but Not in TNF-/- Mice.
TNF can promote the growth of DCs in vitro (15) (16). To assess the role of TNF in the development of DCs in vivo, splenocytes from TNF-/- and wt mice were stained for CD11c and MHC class II (I-Ab), and the number of DCs in the preparation was determined by flow cytometry. The total number of DCs in both types of mice was similar, suggesting that TNF is not essential for the development of DCs (Figure 1 A and Table 1). Interestingly, the number of DCs in LT-/- mice was greatly reduced, especially for the CD11chighclass IIhigh subset (Figure 1 A and Table 1), suggesting a role for LT
in DC development. Soluble LT
and TNF-
are structurally related homotrimers (LT
3 and TNF-
3) that exhibit similar biological activities by binding to the defined TNFRs (1), so TNFR-/- mice were used to determine the role of TNFR in DC development (Figure 1 and Table 1). However, the normal number of DCs in the spleens of TNFR-/- mice suggests that signaling via TNFR by either LT
3 or TNF-
3 is not essential for the presence of DCs in the spleen.
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CD11c+ DC subsets preferentially migrate to distinct areas in the spleen (18) (19): myeloid DCs (CD8-/CD11b+) are mainly located in the marginal zones (MZs) of white pulp, whereas lymphoid DCs (CD8
+/CD11b-) are preferentially located in the T cell zones of white pulp. To study whether LT or TNF preferentially regulates a subset of DCs, the distribution of DCs and B cells in the spleens of TNF-/- mice and LT
-/- mice was visualized histologically (Figure 1 B). Clusters of splenic DCs were readily observed in the T cell zone and MZ of wt and TNF-/- mice; however, only a few dispersed DCs were randomly present in the spleens of LT
-/- mice. The distribution pattern and number of DCs visualized in situ closely correlated to that measured by flow cytometry, which showed that both myeloid and lymphoid DCs were proportionally reduced in LT
-/- mice (Figure 1 C). Considering that myeloid and lymphoid DCs may be distinct populations of DC subsets (18) (19), it is interesting to notice that the presence of both subsets was regulated by LT.
Signaling via LTßR by Membrane LT1ß2 Is Required for the Presence of DCs.
LT-/- mice lack both soluble LT
3 and membrane-associated LT
1ß2, which bind to separate receptors, TNFR and LTßR, respectively (1) (2). As the number of DCs in TNFR-I-/- mice was similar to that in wt mice (Figure 1 A and Table 1), it was possible that membrane LT
1ß2, instead of soluble LT
3, was required for the presence of DCs in the spleen. To test this hypothesis, LTßRIg was used to block membrane LT activity in wt adult mice, which resulted in the absence of FDCs in 1 wk. Interestingly, the number of DCs but not lymphocytes in the spleens was markedly reduced 10 d after the administration of a single dose of LTßRIg (Figure 2 and Table 1). Moreover, the distribution pattern of the remaining DCs in the spleen was similar to that in LT
-/- mice.
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Expression of LT has been detected primarily in activated T, B, and NK cells (1) (2). However, the percentage of DCs in the spleen of TCR-/-BCR-/- CD3-transgenic mice or RAG-1-/- mice is not obviously reduced (data not shown). In fact, the percentage of DCs in the splenocytes of RAG1-/- mice is three- to fourfold higher than that of wt mice (Figure 2a and Figure b). This suggests that the development of DCs could be independent of LT expression on T and B cells. To rule out whether the DC development observed in RAG-1-/- mice might be occurring via an LT-independent pathway, RAG-1-/- mice were treated with LTßRIg for 10 d (Figure 2 and Table 1). A significant reduction of splenic DCs (6090% reduction) was readily detected, demonstrating that LT-expressing cells other than T and B cells control the migration of DCs (Table 1). Although NK cells in RAG1-/- mice were plausible candidates for regulating DC migration in an LT-dependent pathway, RAG-1-/- mice depleted of NK cells (with 300 µg of PK136, an anti-NK1.1 antibody) did not exhibit reduced numbers of splenic DCs. Consistent with this data, no reduction of DCs was detected in CD3
-transgenic mice lacking both NK and T cells. It is likely that cells other than T, B, and NK cells also express low levels of LT, regulating the migration of DCs.
Murine LTßRIg may block ligands other than membrane LT. It has been shown that human LTßRIg can also bind to human LIGHT (homologous to lymphotoxins, exhibits inducible expression, and competes with herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes), a recently identified membrane-associated TNF family member (26). The biological consequence of this binding is unclear. To exclude the potential effect of LIGHT, an antimurine LTß mAb, which specifically binds to the LTß chain but not LIGHT, was administered to wt mice. Such treatment also resulted in a reduced number of DCs and their subsets similar to the effect of LTßRIg (Figure 3 A). Our data clearly indicate that LT1ß2 is the ligand required for the presence of DCs in the spleen. As ligands from the TNF family can bind to more than one receptor, the number of splenic DCs in LTßR-/- mice was determined to directly address whether signaling via LTßR is required for the presence of DCs in lymphoid tissue. The number of DCs in these mice was also lower than in wt mice (Figure 3 B). Thus, the data strongly suggest that signaling via LTßR by membrane LT is essential for the presence of DCs in the spleen.
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Ineffective Migration of wt DCs into Spleens of LT-/- Mice.
Fewer DCs in the lymphoid tissues of mice lacking LT may be related to a reduction of DC progenitors in BM, impaired migration, or an accelerated removal of these cells. To test whether there was a deficiency in DC progenitors or the growth of DCs in LT-/- mice, BM cells from either wt or LT
-/- mice were cultured by standard protocol using different doses of GM-CSF and IL-4 (25). The number of DC colonies and total number of DCs was comparable between wt and LT
-/- mice. In addition, the number of DC colonies from wt mice was not altered by coculture with LTßRIg (data not shown). Together, the data suggest that LT is not an essential survival factor or growth factor for DCs or their progenitors.
It has recently been shown that LT and, to lesser degree, TNF stimulates stromal cells to release chemokines, which may determine the migration or segregation of T and B cells in the spleen (16). It is possible that the migration of DCs into lymphoid tissues of LT-/- mice is impaired due to the lack of LT-mediated chemokines for DCs. If the migration of DCs into lymphoid tissues is impaired in LT
-/- mice, the question would be where DCs accumulate in the absence of LT. If the BMDC development remains functional in the absence of LT, we would expect that the reduced number of DCs in lymphoid tissues in the absence of LT might be associated with an increased number of DCs in nonlymphoid tissues. Interestingly, there is an accumulation of lymphocytes around perivascular areas in lungs, liver, pancreas, submandibular glands, kidneys, and other tissues in LT
-/-, LTß-/-, and LTßR-/- mice (7) (8) (9). To test whether the number of DCs was also increased in nonlymphoid tissues, DCs in lungs were quantitated in wt and LT
-/- mice. In contrast to the reduced number of DCs in lymphoid tissues, the number of DCs in lungs of LT
-/- mice was much higher than in wt mice (10.5 ± 1.8 x 105 vs. 2.9 ± 1.3 x 105). This suggests that LT is required for the proper distribution of DCs.
To directly study whether the migration of DCs into the spleen was impaired in LT-/- mice, DCs expanded from the BM of Ly5.1 wt mice were transferred into LT
-/- and C57BL/6 mice (Ly5.2), respectively. The number of Ly5.1 DCs recovered from the spleens of wt mice was two- to fourfold higher than that from LT
-/- mice, although both groups received similar numbers of DCs from the same source (Figure 4 A). Ly5.1+CD11c- donor cells, mainly macrophages, in both groups were roughly the same (Figure 4 A). As the number of splenic DCs in wt mice was not reduced within the first week after administration of a high dose of LTßRIg, it is unlikely that transfer of Ly5.1 DCs into LT
-/- mice leads to the premature death (<24 h) of these DCs.
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It is possible that the splenic environment in LT-/- mice did not allow the efficient sequestration or migration of DCs. The splenic environment essential for the localization of DCs may include its architecture, the size and shape of white pulps, and cytokines, such as chemokines, produced from the spleen. Altered splenic architecture and smaller white pulp in LT
-/- mice are readily visualized defects that may structurally impair the migration of splenic DCs into the proper area. However, short-term blockage of membrane LT by LTßRIg in wt mice had no detectable impact on the architecture or size of white pulps, yet this treatment still prevented the effective migration of DCs into the T cell zone and B cell follicles (Figure 2 and Table 1). This suggests that altered architecture itself is not the primary cause of reduced migration of DCs into the spleens of LT
-/- mice. Interestingly, the altered T/B cell segregation correlated with the altered localization of DCs (Figure 2) and with altered chemokine production in the absence of LT (16).
To study whether additional membrane LT can restore the localization of DCs in the spleens of LT-/- mice, we transferred LT-expressing lymphocytes and DCs from wt mice into LT
-/- mice. The altered splenic architecture remained, but the number of CD11c+ cells in LT
-/- recipients was comparable to that in wt recipients 10 d after transfer (Figure 4 B), again suggesting that the overall architectural defect in LT
-/- mice may not be the primary cause of reduced number DCs in the spleen. It appears that the microenvironment in the spleen required for the presence of DCs is rather flexible and can be altered in 12 wk. Interestingly, the timing of the reduction of DCs is also consistent with the maximum reduction of various chemokines in the spleen 12 wk after administration of LTßRIg (16). Thus, the data suggest that the reduced number of DCs in LT
-/- mice may be due, at least in part, to the impaired migration of DCs that may be mediated through altered chemokine production. The nature of the LT-responsive stromal cells and the exact type of chemokines remains to be determined.
LT-mediated Microenvironment that Permits the Migration of DCs Is Determined by BM-derived Cells.
BM transfer in long-term reconstitution provides a model to evaluate the role of LT in determination of the splenic microenvironment that permits the migration of DCs. 6 wk after lethally irradiated LT
-/- mice were reconstituted with wt BM, DCs were restored to a level similar to that seen in irradiated wt mice reconstituted with wt BM (Figure 5). This suggests that the altered microenvironment that impairs the migration of DCs is not developmentally fixed and that LT-expressing BM cells could restore the migration of DCs. In contrast, when lethally irradiated wt mice were reconstituted with LT
-/- BM, the number of DCs in the spleen was reduced, as is seen in LT
-/- mice or LTßRIg-treated mice (Figure 5). Therefore, the LT
-mediated microenvironment that permits the migration of DCs is primarily determined and maintained by LT-expressing BM-derived cells.
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Impaired Mixed Leukocyte Reaction in LT-/- Mice.
To examine whether reduced numbers of DCs in lymphoid tissues of LT-/- mice could impair the overall function of DCs, the ability of DCs in LT
-/- mice to stimulate allogenic T cells was evaluated by mixed leukocyte reaction (MLR). Mechanically separated splenocytes from LT
-/- mice showed a decreased ability to stimulate allogenic T cells in a dose-dependent manner (Figure 6 A). To rule out the possibility that reduced antigen-presenting activity in the splenocytes of LT
-/- mice is associated with the failure to release DCs from altered architecture of the spleen using physical separation, spleen fragments from both LT
-/- mice and wt mice were subjected to collagenase digestion to release DCs. The collagenase-treated splenocytes from LT
-/- mice showed profound defects (four- to eightfold lower) in antigen-presenting activity compared with those from wt mice, especially when the total splenocytes was in the range of 0.21 x 105 cells (Figure 6 B). To exclude the impact from either of the developmental defects in LT
-/- mice, the splenocytes from LTßRIg-treated C57BL/6 mice were collected by mechanical pressure and used as stimulators. Severalfold reductions of radiation count were readily detected in the LTßRIg-pretreated group, as in the case of LT
-/- mice (Figure 6 C). In general, the lower MLR closely correlated with the lower number of DCs (Figure 1, Figure 2, and Figure 6). The number of other potential APCs, such as B cells, in the spleens of LT
-/- mice or mice treated with LTßRIg appears to be comparable to that in wt mice. It was proposed a decade ago that DCs are the principal stimulators of MLR in the spleen (27) (28); our results further support the proposal, as reduced numbers of DCs in LT
-/- mice could account for the impaired MLR.
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Our results have revealed that membrane LT and LTßR are the natural ligandreceptor pair essential for the presence of splenic DCs in vivo. LT-/- mice exhibit reduced numbers of DCs in the spleen, whereas both TNF-/- and TNFR-/- mice show normal numbers of splenic DCs, suggesting that signaling via TNFR by either soluble LT
or TNF is not an essential pathway for the regulation of DCs in the spleen. The notion that membrane LT is an essential ligand for the presence of DCs in the spleen is further supported by the reduced number of DCs in the wt spleen after the administration of either LTßRIg or anti-LTß mAb. The results also suggest that signaling via LTßR by membrane LT is required for the presence of DCs, as LTßR is the only identified receptor for membrane LT. Finally, the lower number of splenic DCs in LTßR-/- mice confirms our hypothesis. In terms of the regulation of development or migration of DCs in the spleen, an essential role of either soluble LT
3 or TNF-
3 has not been demonstrated. However, TNF-
3 or LT
3 can coordinate membrane LT
1ß2 in the development of lymphoid tissues (2) (10) and also may play a minor role in the migration of DCs in some situations. Interestingly, recent studies reported that high levels of soluble LT
3 were able to induce chemokines and adhesion molecules in vitro (29). Ectopic expression of LT
3 induces lymphocyte infiltration in nonlymphoid tissue, suggesting that the overexpression of LT
3 may still play a role in the migration of some lymphoid cells (30) (31) (32). Ectopic LT in LT
-/- (RIPLT.LT
-/-) mice also restored some LN, but a decreased number of interdigitating DCs was apparent in the LN (31). Therefore, proper expression of LT in the LN may also be required for the presence of DCs in the LN.
The ineffective migration of DCs may account for the reduced number of DCs in the spleens of mice lacking membrane LT or its receptor: (a) compared with wt recipients, fewer donor DCs were present in the spleens of LT-/- recipients; (b) a reduced number of DCs is not developmentally fixed and can be repaired by LT-expressing cells; (c) the timing of altered numbers of DCs is consistent with the altered expression of chemokines in the spleen; (d) no significant impairment of DC growth or reduced DC progenitors can be detected; and finally, (e) DCs accumulate in nonlymphoid tissues in both LT
-/- and LTßR-/- mice, strongly supporting our notion that the reduced number of DCs in the spleen is caused by impaired migration. Interestingly, fewer randomly distributed DCs in the spleens of LTBRIg-treated mice could still move to the T cell zone after intravenous injection of LPS, suggesting that fine positioning of DCs in the spleen could be regulated in an LT-independent fashion.
A number of chemokines are constitutively secreted in the lymphoid organs in an LT-dependent fashion (16). Altered distribution of T cells, B cells, and DCs in vivo may be regulated by some chemokines. Whether proper distribution of DCs and FDCs will facilitate T/B cell segregation remains to be determined. Although the expression of several chemokines has been found to be downregulated in the absence of LT, the exact chemokine that is essential for the migration of DCs has yet to be identified. Which chemokines are upregulated for directing DCs into nonlymphoid tissues in the absence of LT is completely unknown. Interestingly, the migration of most subsets of macrophages in the spleen is largely unchanged in the absence of LT (Figure 4 A), suggesting that the chemokines that regulate the distribution of DCs may be distinct from those that regulate the distribution of macrophages. It will be important to determine whether the differences in the migration patterns of macrophages and DCs may account for differences in their biological activities. In addition to the action of LT on stromal cells, it is also possible that direct signaling via LTßR on DCs by membrane LT is required for the migration of DCs in the spleen.
Reduced numbers of DCs may account for reduced MLR, which is a DC-based T cell response. However, migration of DCs into lymphoid tissues for systemic immune responses may be more important for the generation of immune responses in vivo. In fact, after capturing antigens outside lymphoid tissues, DCs must migrate into lymphoid tissues to prime rare antigen-specific lymphocytes, which constantly recirculate through peripheral lymphoid tissues (18) (19). Regulation of the migration of DCs may provide an additional means to manipulate immune responses, T cell responses in particular. Consistent with that notion, we have found that inhibition of membrane LT has profound effects in several T cellbased disease models. For example, administration of LTßRIg reduced severity of colitis (17), collagen-induced arthritis, and experimental autoimmune encephalitis (J.L. Browning, unpublished observation). Clearly, the membrane LT/LTßR system provides an interesting model to further study DC biology and DC-mediated diseases.
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Acknowledgements |
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The authors gratefully acknowledge the technical assistance of Guangming Huang and generous support of Dr. David Chaplin. The authors would like to thank Drs. Yong-Jun Liu, Godfrey Getz, Don Rowley, and Hans Schreiber for their critical comments and advice.
This work was supported in part by grants AI01431, HD37600, and HD37104 from the National Institutes of Health, and grant RG3068-A from the National Multiple Sclerosis Society (all to Y.-X. Fu).
Submitted: 26 March 1999
Revised: 21 June 1999
Accepted: 22 June 1999
1used in this paper: BM, bone marrow; DCs, dendritic cells; FDCs, follicular dendritic cells; LT, lymphotoxin; MLR, mixed leukocyte reaction; MZs, marginal zones; wt, wild-type
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References |
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