Differential regulation of CCL21 in lymphoid/nonlymphoid tissues for effectively attracting T cells to peripheral tissues (original) (raw)
Preferential accumulation and homing of T cells to the lungs of LT-deficient mice. Earlier gene targeting studies of the LT pathway revealed a spontaneous accumulation of immune cells in the lungs of LTα–/– and LTβR–/– mice, the basis of which remains unknown (21, 26). In order to further understand the nature of the inflammatory infiltrate in LTα–/– mice, we determined the cellular composition of immune cells in the lungs of WT and LTα–/– mice (Figure 1a). Consistent three- to fivefold increases in the number of CD4+ T cells, B cells, and DCs were detected in the lungs of LTα–/– and LTβR–/– (data not shown) compared to WT mice (Figure 1a). A more modest twofold increase in CD8+ T cells was also observed in LTα–/– lungs (data not shown). The increase in CD4+ T cells, B cells, and DCs in LTα–/– lungs is specific as NK cells and macrophages are not increased (Figure 1a; data not shown). Interestingly, the three cell types overrepresented in the LTα–/– lung are all known to express the CCR7 chemokine receptor and exhibit chemotactic activity towards its ligand CCL21 (27–31). This raises the possibility of altered CCL21 expression in the lung, leading to the accumulation of leukocytes in the lung.
Leukocyte accumulation in lungs of LT-deficient mice. (a) Increased numbers of CD4+, B220+, and DCs in lungs of LTα–/– mice. Data are mean ± SD of triplicate samples and represent at least three independent experiments. (b) CCR7 expression on naive and activated phenotype pulmonary CD4+ T cells. Representative dot plots and histograms of CCR7 chemokine expression using ELC-Fc (blue lines) compared to control human-Fc (red lines) on CD4+CD62Lhigh and CD4+CD62Llow cells. (c) Altered homing of CD4+ T cells to the lungs of LTβR–/– mice. The spleens and lungs of WT and LTβR–/– mice were analyzed for the presence of OTII cells 2 days after adoptive transfer i.p. of Thy1.1+ OTII cells. Data are mean ± SD of four or more mice per group. *P < 0.05 between WT lung and WT spleen, and WT lung and LTβR–/– lung as calculated by the Student’s t test. Max, maximum.
The increased numbers of leukocytes in the lungs of LT-deficient mice may reflect a selective accumulation of naive or activated cells. To better characterize the nature of the lymphoaccumulation, flow cytometry analysis was performed to determine the activation status of the T lymphocytes in WT and LTα–/– lungs. The analysis revealed that the lymphoaccumulation includes both activated (CD62Llow) and naive (CD62Lhigh) phenotype CD4+ T cells in the lungs of LTα–/– mice (Figure 1b). Staining with the activation marker CD44 also showed that both antigen-experienced and naive phenotype CD4+ T cells accumulate in the lungs of LTα–/– mice. Additionally, we found a slight increase in activated B cells and mature DCs in the lungs of LTα–/– mice (data not shown). To test our hypothesis that CCL21 expression in the lung was attracting CCR7 expressing T cells, ELC-Fc was used to determine if CCR7+ cells are present in the lung. We found that both naive and activated phenotype CD4+ T cells express CCR7 (Figure 1b). These data indicate that both CCR7+ activated or naive T cells accumulate in lungs of LT-deficient mice and suggest that CCL21 may be responsible for attracting the cells via CCR7.
The lymphoaccumulation observed in the lungs of LT-deficient mice may be a result of enhanced homing to the lung or increased cell proliferation. To directly interrogate the role of altered homing, antigen-activated WT OTII CD4+ TCR transgenic T cells bearing the Thy1.1 congenic marker were adoptively transferred into WT or LTβR–/– mice expressing Thy1.2. The spleen and lung were analyzed for the presence of OTII cells 40 hours after adoptive transfer. CD4+ OTII cells preferentially homed to the spleen and significantly lower to the lung in WT mice (P < 0.05 between WT spleen and lung using the Student’s t test) (Figure 1b). By contrast, the homing profile of CD4+ OTII cells was perturbed in LT-deficient mice with robust homing to the lung that was on par with the spleen and significantly greater than WT lung (P < 0.05 between LT-deficient and WT lungs using the Student’s t test) (Figure 1b). Increased homing to the lungs of LT-deficient mice included naive OTII cells (data not shown). These results indicate that CD4+ T cells preferentially home to the lung in LT-deficient mice and suggest that increased homing is a major mechanism for the accumulation of leukocytes in the lung.
Lymphotoxin regulates splenic chemokine expression. We hypothesized that increased expression of chemokines in the lung or an altered balance between the spleen and lung of LT-deficient mice was responsible for enhanced cell homing to the lung. To unravel the relationship between LT and chemokines in a systematic and unbiased fashion, we studied chemokine expression in LT-deficient mice using gene array analysis. Splenic and lung RNA from naive WT and LTα–/– mice were extracted and subjected to a chemokine gene array. Our analysis revealed dramatic reductions in the chemokines SLC/CCL21, RANTES, IFN-γ-inducible-10 (IP-10)/CXCL10, BLC/CXCL13, and stromal cell–derived factor-2 (SDF-2) in the spleens of LTα–/– mice (Figure 2a). The expression of numerous other chemokines, including thymus and activation-regulated chemokine/CCL17, CCL6, interferon inducible-T cell alpha chemoattractant/CXCL11, CXCL15, macrophage inflammatory protein-2, and SDF-1α/CXCL12 was comparable between WT and LTα–/– spleens (Figure 2a). Our analysis of the lung revealed similar chemokine profiles between WT and LTα–/– mice and, importantly, that none of the chemokines were significantly elevated in LT-deficient mice (Figure 2b). These data indicate that LT is required for the basal expression of a specific subset of chemokines in the spleen but not in the lung.
Lymphotoxin regulates splenic chemokine expression. (a, b) RNA from WT and LTα–/– spleen (a) and lung (b) were subjected to chemokine gene array. All signals were normalized to β-actin, which was set to 10,000. Asterisks indicate that no signal above background was detected. RNA from at least three mice was pooled together. A representative blot from two independent experiments is shown. (c, d) LT regulates splenic but not pulmonary CCL21. OCT-embedded (Sakura Finetek, Torrance, California, USA) spleen (c) sections (×100) were stained for CCL21 (blue) and B220 (brown). Formalin-fixed lung (d) sections (×200) were stained for CCL21 (red) and counterstained with hematoxylin. A representative staining from more than five experiments is shown. TARC, thymus and activation-regulated chemokine; I-TAC, IFN-inducible T cell α-chemoattractant; MIG, monokine induced by IFN-γ; MIP, macrophage inflammatory protein.
An alternate hypothesis was that the distorted localization of chemokines or imbalanced chemokine gradient between the spleen and lung in LT-deficient mice is responsible for the perturbed homing. The chemokine CCL21 piqued our interest because genetic studies examining LTα–/–, plt/plt (both with dramatically reduced lymphoid tissue CCL21) and CCR7–/– (CCL21 receptor) mice revealed a critical role for CCL21 in T cell homing to lymphoid organs (9, 10). Additionally, plt/plt or WT mice treated with anti-CCL21 antibodies paradoxically mounted a delayed but exaggerated peripheral immune response (32, 33). To visualize the expression pattern of CCL21 in the tissues, we performed immunohistochemistry staining for CCL21 on the spleens and lungs of WT and LTα–/– mice. CCL21 staining was noticeably absent in the LTα–/– spleen, in contrast to the abundant staining seen in the T cell zones of WT spleen (Figure 2c). LT deficiency, however, had no effect on the pattern of CCL21 staining in the lung (Figure 2d). The immunohistochemistry staining confirmed the essential role of LT for CCL21 expression in the spleen but not in the lung.
Baseline CCL21 expression in the lung is independent of LT, lymphocytes, p52, and RelB. We propose that LT may not positively regulate CCL21, a CCR7 ligand, in the lung and that LTα–/– mice may have similar or increased levels of CCL21 in the lung that is responsible for the accumulation of T cells, B cells, and DCs. To test this hypothesis, we performed an ELISA for CCL21 from splenic and lung homogenates of WT and various LT-deficient mice for a more quantitative assessment of actual protein levels (Figure 3a). In the spleen, LTα–/– mice displayed more than a 20-fold reduction in CCL21 (Figure 3a). Since LTα–/– lacks both soluble and membrane forms of LT, we examined LTβ–/– to dissect the contribution of membrane LT signals. LTβ–/– mice exhibited a similar defect in CCL21 expression in the spleen but not in the lung (Figure 3a). To define whether LTβR is essential for the expression of CCL21 in the spleen and lungs, we analyzed LTβR–/– mice. Similar to LTα–/– and LTβ–/– mice, LTβR–/– mice have very low levels of CCL21 in the spleen, confirming that signaling of LTβR by membrane LT is required for proper expression of CCL21. However, no significant reduction of CCL21 in the lung was found in the LT-ligand and receptor-deficient mice, leading to a reversal of the WT situation with relatively high levels of CCL21 in the lungs of LT-deficient mice. The data demonstrate that, in stark contrast to the spleen, LT is not required for the constitutive expression of CCL21 in the lung.
CCL21 is differentially regulated in the spleen and lung. (a) LT signals are essential for spleen but not lung CCL21. ELISA for CCL21 was performed on spleen and lung homogenates prepared from various LT-deficient mice. Each sample was normalized by tissue weight. (b) Differential requirements for NF-κB and lymphocytes in the expression of CCL21 in the spleen and lung. ELISA for splenic and lung CCL21 from various NF-κB–deficient and Rag–/– mice, calculated as in a. Each symbol represents data from a single mouse. (c, d) LT selectively regulates the expression of CCL21-Ser but not CCL21-Leu. Real-time PCR analysis was performed on the spleen and lung of WT and LTα–/– mice with conditions specific for CCL21-Ser (c) and CCL21-Leu (d). The columns and bars represent the mean ± SD.
Recent reports have shown that LT utilizes the noncanonical p52/RelB NF-κB pathway to induce chemokine expression (3, 25). To exclude the possibility that other ligands or factors in the lung may be compensating for LT to activate p52 and RelB, we quantified CCL21 expression in the lungs and spleens of p52–/– and RelB–/– mice (Figure 3b). Our analysis again revealed a similar segregation between the spleen and lung: that p52–/– and RelB–/– mice had approximately a 10-fold reduction in CCL21 in the spleen without appreciable loss in the lung (Figure 3b). Finally, we tested for the requirement of lymphocyte-derived factors in the maintenance of CCL21 in the lung (Figure 3b). Although lymphocyte-derived factors are essential for splenic CCL21 expression, lymphocytes are not required for the expression of CCL21 in the lung because WT and Rag–/– mice had similar levels of pulmonary CCL21 (Figure 3b). These results indicate that constitutive expression of CCL21 in nonlymphoid tissues such as the lung is independent of lymphocytes, and the LT and p52/RelB pathways.
Lymphotoxin selectively regulates CCL21-Ser. B6 mice express two different CCL21 genes, but the role of LT in the regulation of each of these genes has not been well defined. The abundant expression of CCL21 in nonlymphoid tissues of LTα–/– mice suggested to us that an LT-independent CCL21 gene is predominantly expressed in nonlymphoid tissues. To formally address this issue, real-time PCR analysis with probes specific for each of the two CCL21 genes was performed on RNA extracted from the spleens and lungs of WT and LT-deficient mice. The abundant CCL21-Ser transcripts in the WT spleens were conspicuously absent in the LTα–/– spleens (Figure 3c). In contrast, the absence of LT did not alter the transcriptional activity of CCL21-Ser in the lung and CCL21-Leu in the spleen or lung (Figure 3d). In addition, we cloned and sequenced the expressed CCL21 gene products in the spleen and lung by RT-PCR with primers that do not discriminate between the known CCL21 genes. Impressively, 11/11 clones in the spleen carried the serine at position 65, whereas 4/4 clones derived from the lung coded for leucine at position 65, confirming the results from real-time PCR. Furthermore, in vivo administration of agonistic anti-LTβR, but not control antibodies, selectively stimulated the CCL21-Ser gene in both the spleen and lung (Figure 4a). The effects of LTβR signaling were specific to CCL21-Ser, since CCL21-Leu transcriptional activity was not affected in the spleen and lung (Figure 4a). These data indicate that LT selectively regulates CCL21-Ser but not CCL21-Leu.
Airway inflammation induces the CCL21-Ser gene in an LT-dependent fashion. (a) LTβR signaling specifically induces CCL21-Ser. WT mice were injected i.p. with control or agonistic LTβR antibodies. RNA from the spleens and lungs were analyzed by real-time PCR for CCL21-Ser and CCL21-Leu. Each point reflects the fold induction in CCL21. (b) Spleen and lung cells respond to LTβR signals. Representative histograms of stromal cells from the spleen or lung stimulated with species control (black) or agonistic anti-LTβR (gray) antibody and stained with VCAM-1 antibody for analysis by flow cytometry is shown. (c) Airway inflammation induction of CCL21 in the lung is LT-dependent. WT or LTα–/– mice were challenged with SEA and control or anti-LTβ antibodies were administered i.p. during the challenge phase only. Mice were killed 3 to 4 days after challenge and pulmonary CCL21 quantified as previously described (see Figure 3a). Student’s t tests were performed between naive and challenged, control challenged and anti-LTβ–challenged, and WT and LTα–/– SEA-challenged groups and the resultant P values are shown. (d) Airway inflammation specifically induces the CCL21-Ser gene. Real-time PCR for the two CCL21 genes was performed on RNA extracted from lungs of naive and SEA-challenged WT and LTα–/– mice. PCR reactions were performed in duplex with a GAPDH internal control for normalization. Columns and bars represent the mean ± SD.
Airway inflammation specifically induces CCL21-Ser in the lung in a LT-dependent fashion. To formally demonstrate that spleen and lung cells express the LTβR and functionally respond to LT signals, we isolated splenic and lung stromal cells and tested their ability to respond to agonistic anti-LTβR or hamster control antibodies. We found that both splenic and lung stromal cells responded to LTβR signals by inducing the adhesion molecule VCAM-1 (Figure 4b). This induction was specific for the LTβR, since LTβR–/– stromal cells, regardless of splenic or lung origin, did not respond to the anti-LTβR antibody. Furthermore, CCL21 could be induced on embryonic fibroblasts 8 hours after stimulation with the agonistic antibody (data not shown). This demonstrates that spleen and lung cells not only express the LTβR but are capable of responding to membrane LT signals. Therefore, we proposed that the paucity of LT-expressing cells in the lungs of unchallenged mice may not allow for strong stimulation of the CCL21-Ser gene.
To test our hypothesis, we sought to recruit LT expressing cells to the lung in a model of airway inflammation to further induce pulmonary CCL21 expression via CCL21-Ser. An S. mansoni SEA airway hypersensitivity model was chosen for its ability to induce a strong immune response in the lung. SEA challenge significantly induced pulmonary CCL21 expression, about two- to threefold above unchallenged mice (P = 0.0005 in the Student’s t test between naive and challenged groups) (Figure 4c). To determine which of the CCL21 genes is responsible for the increased expression upon airway challenge, real-time PCR analysis was performed to distinguish between the two CCL21 genes. We found that an SEA challenge specifically induces the transcriptional activity of CCL21-Ser by approximately sixfold, whereas no significant induction was detected in CCL21-Leu (Figure 4d). Since the activity of CCL21-Ser is regulated by LT in the spleen, we assessed the role of LT in pulmonary CCL21-Ser induction. To this aim, we used antagonistic anti-LTβ or control antibodies during the challenge phase only in order to not disturb priming in our SEA model. This anti-LTβ treatment significantly reduced the levels of CCL21 from the control-challenged group (P = 0.0009 between SEA-challenged and anti-LTβ-treated groups). Impressively, blockade of membrane LT only in the challenge phase abolished the induction in CCL21 to preairway challenge levels (P = 0.51 between naive and SEA-challenged anti-LTβ treated groups) (Figure 4c). As further support, SEA-challenged LTα–/– mice failed to induce CCL21 in the lung and had significantly lower levels of CCL21 than SEA-challenged WT mice (P = 0.013 between SEA-challenged WT and LTα–/–), confirming the requirement of LT in pulmonary CCL21 induction (Figure 4c). These results demonstrate that chemokines such as CCL21 can be further induced in the lung under local inflammatory conditions and that this may serve to further stimulate the migration and homing of effector cells.
Homing of T cells to the lung requires CCR7 and CCL21. Although CCL21 has been shown to attract activated T cells in vitro, its role in vivo for primed T cells remains to be understood (27, 28, 34). In order to ascribe a role for CCL21/CCR7 interactions in cell migration to the lung, we took advantage of the inflammatory leukocytes in the lungs of SEA-challenged mice for adoptive transfer to naive mice. We reasoned that because T cells from the lungs of SEA-challenged mice have already homed to the lung, they should therefore possess all the homing receptors necessary for entry to the lung. The T cells from SEA-challenged mice are predominantly CD4+ Th2 phenotype cells that have acquired an activated phenotype (CD62Llow) and express the CCR7 receptor (Figure 5a). In addition, the majority of the T cells also stain CD44high (data not shown). Inflammatory leukocytes were isolated from lungs of SEA-challenged WT mice, fluorescently labeled with CFSE and Thy1.2-PE antibodies, treated with either PBS or a high dose of CCL19 to desensitize the CCR7 receptor prior and adoptively transferred to naive LTα–/– recipient mice. LTα–/– mice were used as recipients rather than WT mice, which have relatively higher expression of CCL21 in lymphoid tissues that may preferentially divert homing away from the lung. To rule out nonhoming-related effects, such as further differentiation or activation, and to avoid CCR7-resensitization, LTα–/– recipient mice were killed 1 hour after transfer to determine the number of T cells that homed back to the lung. The mice were bled and perfused to eliminate leukocyte contamination from the circulation. T cells (Thy1.2+CFSE+) from the CCR7-desensitized group significantly decreased homing to the lung than control T cells, whereas non-T cells (Thy1.2-CFSE+) did not seem to be affected by CCR7 desensitization (P < 0.05 between T cells from CCR7-desensitized and control groups) (Figure 5b). Furthermore, lung sections from the CCR7-desensitized group exhibited approximately a threefold reduction in the number of Thy1-PE antibody–labeled cells compared with the control group and confirmed their localization to the lung tissues. These results demonstrate for the first time the importance of CCR7 on the T cell for homing to nonlymphoid tissues.
CCL21 and CCR7 on the T cell are required for homing to the lung. (a) Phenotypic characterization of T cells from lungs of SEA-challenged mice used in adoptive transfer experiments. Representative staining for CD4 and CD62L is shown. The numbers adjacent to the gates indicate the percentage of cells within the gates. A representative histogram is shown for CCR7 expression on gated CD4+ T cells using control human-Fc (red) and ELC-Fc (blue). (b) The CCR7 chemokine receptor on the T cell is required for homing to the lung. Lung cells from SEA-challenged mice were prelabeled with Thy1.2-PE and CFSE, treated with PBS or CCL19 to desensitize the CCR7 receptor, and adoptively transferred i.v. to naive LTα–/– mice. Recipient mice were killed 1 hour later, and the lungs were collected for analysis. Leukocytes were isolated from the lungs and subjected to flow cytometry analysis. Representative dot plots from control, CCR7 desensitized groups, and cells prior to transfer are shown. The numbers next to the gates indicate the percentage of cells within the gate. (c) CCL21 is required for T cell homing to the lung. Recipient mice were pretreated 1 hour prior to adoptive transfer with anti-CCL21 or control antibodies. Lung leukocytes were extracted 4 hours after adoptive transfer, and the T cells were enumerated. *P < 0.05 as calculated from Student’s t test. Data are pooled from at least four independent experiments.
The foregoing results suggested that CCL21, a chemokine for CCR7 in the lung, mediates T cell homing to the lung. CCR7, however, binds to both CCL19 and CCL21. We did not believe that the effects of CCR7 desensitization were attributed to CCL19, since it was neither detectable by Northern blot (35) nor by sensitive ELISA (data not shown) in the lungs of the recipient mice, whereas CCL21 is readily detectable (Figure 3a). To directly dissect the role of CCL21 in T cell homing to the lung, we pretreated the recipient mice with neutralizing anti-CCL21 or control antibodies prior to adoptive transfer of labeled inflammatory leukocytes from the lung. Neutralization of CCL21 dramatically decreased the number of T cells that homed to the lung after 4 hours (P = 0.03 between the two groups) (Figure 5c). These results indicate that CCL21 is essential for T cell homing to the lung.