Pulmonary Stromal Cells Induce Immature Dendritic Cells To Differentiate Into Regulatory Dendritic Cells

Dendritic cells (DCs) are the most potent antigen-presenting cells (APC) in the immune system, with unique capacity to activate naive T cells and subsequent initiate the immune response. Now, more and more evidences demonstrate the heterogeneity of DCs. DCs can not only initiate but also tolerize immune response. There are both immunogenic DCs and tolerogenic DCs in the immune system. As to the studies of tolerogenic DCs or regulatory DCs with functions to negatively regulate the immune response, most of researchers used immunosuppressive cytokines such as IL-10, TGF-β or immunomodulatory drugs to culture DCs progenitors or immature DCs and induced generation of tolerogenic DCs or regulatory DCs, however, the concentrations of the immunosuppressive cytokines used in the co-culture system are far above the physiologic concentration in vivo, thus the results can not represent the physiologic conditions of tolerogenic DCs or regulatory DCs in vivo.Organism is a complex system consisting of many different organs or systems. The immune system is necessarily influenced by other systems such as respiratory system or gastric system to adapt itself more harmoniously with the organism. Our previous work showed that splenic stromal microenvironment plays a pivotal role in the development of regulatory DCs from hematopoietic progenitor cells and mature DCs. Splenic stromal cells can not only program hematopoietic stem cells to differentiate to regulatory DCs but also drives mature DCs to proliferate and differentiate into regulatory DCs. It seems that lymphoid organ microenvironment plays an important role in immune tolerance. Therefore, we are wondering what's therole of the stromal microenvironment in other nonlymphoid organs such as lung, liver and brain in the immune regulation or immune tolerance?Lung is one of the largest organs connecting with environment. The respiratory mucosa interfaces broadly with the outside environment and therefore the immune cells in lung encounter a vast quantity of environmental antigens, so, it seems tolerance is especially important to respiratory system. Stromal cells, acting as the structure component, not only fulfill the structural function but also are modulators of the immune cells. It has been shown that lung fibroblast can inhibit activation-induced death of T cells through PGE2-dependent mechanisms. So, our hypotheses is that pulmonary stromal cells may influence the development and functions of DCs. Considering the complexity of primary cultured stromal cell, we established a murine fibroblast-like pulmonary stomal cell line (MPSC) and studied the influences of MPSC on the development and functions of DCs.At first, we isolated the lungs of new-born mice, chopped the lungs into small pieces, placed the pieces on the well bottom of 24-well plate and cultured them in RPMI 1640 medium supplemented with 20%FCS. With the continual passages for about 4 months, we got a fibroblast-like stromal cell line and designated it as murine pulmonary stromal cells (MPSCs). MPSCs were in spindle-like shape under microscope, and abundant in ribosomes (R), endoplasmic reticulum (ER) and Golgi apparatus (Go) under transmission electron microscope. MPSCs were vimentin positive, while cytokeratin and desmin negative. MPSC expressed CD105, CD106, MHC class I, while did not express MHC class II, CD31, vWF, CD40, CD54. The results of RT-PCR and ELISA revealed MPSC secreted high level of M-CSF, TGF-6, VEGF, SCF, MCP-1, IP-10 and SDF-la/B. So we characterized it as fibroblast-like stromal cells.Considering that DCs residing in peripheral organ are immature DCs, we used CDllc+DCs derived from bone marrow (BM) after culture for 7 days. BM-derived CDllc+DCs (BM-DCs) cultured with GM-CSF(10ng/ml) and IL-4(lng/ml) for 7 days expressed low level of la, CD40, CD80, and CD86, exhibited a strong endocytic ability, thus generally considered as immature DCs. So, we selected the d7 BM-DCs to mimic the in vivo counterpart immature DCs residing in lung. After seeding immature DCs on the MPSC monolayer, we found that MPSC supported the proliferation of immature DCs. To exclude the contamination of other cells, we sorted single d7 CDllc+IalowDCs from BM-DCs of GFP transgenic mice (GFP+CDllc+IalowDCs) into wells of 96-well plates pre-cultured with MPSC monolayer and examined the proliferation of DCs. We found 90% of the sorted cells proliferated robustly, thus convincingly demonstrating that MPSC indeed supported the proliferation of immature DCs rather than other cells.To observe the dynamic changes of immature DC seeded on the MPSC monolayer, we used GFP+ immature DCs which could be easily distinguished from MPSC in the co-culture system. After co-culture with MPSC, the expression of la and CDllc on DC declined, while the expression of CDllb and CD80 increased, but expression of CD40 and CD86 remained unchanged. Finally, the generated DC showed a phenotypic characteristic of CDllclowfatowCDllblli811. As MPSC did not express CDllb, we selected CDllb as the marker of CDllclowIalowCDllbhieh DC to purify the DC from the co-culture system.To characterize the DC generated from the co-culture system, we compared immature DC (d7 BM-derived DC) with CDllclowIalowCDllbhighDC generated from the co-culture system. The CDllclowIalowCDllbhighDC were tolerogenic to LPS stimulation, showing no difference in phenotype, cytokine secretion and Tcell-stimulating capacity before and after LPS stimulation, while immature DC were sensitive to LPS stimulation, showing rapid changes from immature state to mature state. The CDllclowIaIowCDllbhighDC exhibited low endocytic ability, secreted high level of IL-10, NO, and PGE2, and low level of IL-12, and stimulated poor proliferation of allogenic T cells. Though the CDllc^Ia^CDllb^DC stimulated poor proliferation of allogenic T cells, they could enhance the expression of activation markers CD25 and CD44 on T cells.On the basis of the above results, we proposed that the CDllclowIalowCDllbhighDC generated from co-culturing with MPSC might play a role in the negative regulation of immune response. In order to confirm our hypothesis, we added the CDHclowIalowCDllbhighDC to the OVA323-339 antigen-specific CD4+T/mature DC/OVA323-339 co-culture system, and found the CDllclowIalowCDllbhighDC could potently inhibit the proliferation of antigen-specific T cells induced by mature DC. From the absolute T cell number to CFSE labeled proliferation we detected, we proved the inhibitory role of the CDllc^Ia^CDllb^DC in CD4+ T cell proliferation, so we designated the CDllcl0WIal0WCDllbhighDC as regulatory DC (DCreg). By detecting the cytokines in the co-culture supernatants, we unexpectedly found that the same or even higher levels of IL-2 and IFN-y were in the supernatants of DCreg/CD4+T/maDC/OVA323-339 when compared with that in the supernatants of CD4+T/maDC/OVA323-339. To exclude the possibility of cytokine secretion by DC, by using intracellular staining, we found DCreg didn't inhibit T cell IL-2 and IFN-y secretion activated by mature DC. Furthermore, we demonstrated that DCreg didn't inhibit the expression of activation marker CD25 and CD44 on T cells induced by mature DCs.Next, we studied the mechanisms underlying the differentiation of immature DCinto DCreg induced by MPSC. We wanted to know whether the differentiation was cell-cell contact-dependent or soluble factor-dependent. As immature DCs exhibit strong endocytic ability, we could not use fixed MPSC, so, we used transwell system to study the mechanisms. We added immature DC into the upper or lower chambers of the transwell, with MPSC cultured in the lower chamber. 7 days later we compared the number and function of recovered DCregs. We found the number of recovered DCregs from the upper chamber was far lower than that of lower chamber, while both recovered DCregs could inhibit the proliferation of T cells induced by mature DCs, indicating that proliferation of immature DCs on MPSC is cell-cell contact-dependent, while the differentiation of immature DCs into DCregs is cell-cell contact independent but soluble factors-dependent. To clarify the factors involved in differentiation of immature DCs to DCregs, we added neutralizing antibodies of VEGF, TGF- P and M-CSF to the MPSC/immature DC co-culture system respectively, and then observed the inhibitory function of DCregs generated from the co-culture system in the presence of neutralizing antibodies. We found the the capacity of DCregs to inhibit T cell proliferation remained unchanged, indicating that all of VEGF, TGF- P and M-CSF are not involved in the differentiation of immature DC into DCreg.It was reported lung fibroblasts secreted PGE2, and PGE2 could drive human immature DC to differentiate into regulatory cells in vitro. So, we examined the PGE2 secretion of MPSC and found MPSC secreted PGE2 indeed. Therefore we explored the role of MPSC-derived PGE2 in the differentiation of immature DC into DCreg. We added cyclooxygenase (COX) 1 and 2 nonspecific inhibitor indomethacin to the MPSC/immature DC co-culture system, and studied the inhibitory function of DCreg 7 days later. We found the indomechacin didn't affect DCreg-mediated inhibition of T cell proliferation, indicating PGE2 did not play any role in the differentiation ofDCreg generated by co-culturing with MPSC. Other factors or unknown factors which drive the differentiation of immature DC into DCreg need further investigation.To explore the mechanisms underlying the inhibition of T proliferation by DCreg, we used transwell system as well. We found the DCreg-mediated inhibition of T cell proliferation was cell-cell contact independent but soluble factors-dependent. As DCreg secreted high level of immunosuppressive IL-10 and TGF- P , so, the neutralizing antibodies of IL-10 and TGF- 0 were added to theDCreg/CD4+T/maDC/OVA323-339 system. We found either factor played a role in the inhibition of T proliferation by DCreg. Next, we examined the roles of PGE2, IDO and NO in the inhibition of T proliferation by DCreg. The inhibitors PBIT, 1-methyltryptophan, indomethacin were added to theDCreg/CD4+T/maDC/OVAj23-339 system respectively and the T cell numbers were counted 5 days later. We found the three factors were all involved because the T cell numbers were all elevated in the co-culture system in the presence of these three kinds of inhibitors, with most significant when indomethacin was used. These data demonstrated that PGE2, IDO, and NO are all involved in the DCreg-mediated inhibition of T cell proliferation, and PGE2 is more potent than the other two.It has been shown that DCreg can induce T cell anergy and generation of suppressive or regulatory T cells, which are the main mechanisms by which DCreg exert their inhibitory function. We cocultured GFP+OVA323-329-specific CD4+ T cells with DCreg in the presence of OVA, and purified T cells 7 days later. The co-cultured GFP+T cells were restimulated with mature DC and OVA323-329 or added to the GFP'CD4+T/mature DC/OVA323-329 system. We found the GFP+T cells proliferated poorly under the restimulation and also inhibited the proliferation of GFPT cells. The results indicated that DCreg induced both hyporesponsiveness of T cells andgeneration of regulatory T cells.To characterize the regulatory T cells induced by DCreg, we examined the cytokine expression and phenotype of the T cells generated from the above co-culture system. The T cells secreted high level of TGF- P , IL-10, IFN- Y but negligible IL-4 and IL-2. Using transwells and neutralizing antibodies to TGF- P and IL-10, we found the inhibition of T cell proliferation by regulatory T cells was cell-cell contact-independent and IL-10 or TGF- 3 independent. Thus regulatory T cells generated from co-culturing with DCregs are different from conventional Trl and Th3.In order to investigate the physiological significance of DCreg we observed in vitro and to exclude the possibility of artifical results, we should answer the question that is there natural counterpart of the DCreg (CDllclowIalowCDllbhighDC in vitro generated by co-culturing with MPSC) in lung. Therefore, we digested the lung tissue and analyzed the phenotype of lung mononuclear cells. Considering the CDllc^Ia^CDllb111811 phenotypic characteristic of DCreg, we labeled the lung single mononuclear cells with CDllb-FITC, CDllc-PE-Cy7, Ia-PE, and found there was a cell subset characterizing CDllb^CDllc^Ia10*, which accounted for 4.2% of the total lung CD4"CD8"B220~Gr-l"NKl.l"mononuclear cells. Then we sorted the subset, and analyzed whether the subset also exhibited the inhibitory effect on T cell proliferation. We found that the ex vivo CDllbhighCDllclowIalow cells inhibited T cell proliferation to about 50%. Thus we successfully identified the natural and functional counterpart of DCreg in lung.To clarify the in vivo role of DCreg in the negative regulation of T cell response or T cell-mediated inflammation, considering DCreg induced IFN- y secretion by naive T cells and inhibited IL-4 and IL-10 rather than IL-2 and IFN- Y secretion by Tcells stimulated with maDC, we prepared the mouse model of experimental allergic asthma, with Th2 lymphocyte-mediated airway eosinophilic inflammation. We transferred the DCreg cultured in vitro to the recipients immunized with OVA 24 hours before challenge. We found the mice injected with DCreg developed less severe inflammation as compared with mice injected with immature DC, with lower level of total lymphocytes, eosinophils, IL-4 and IL-5 in the bronchoalveolar lavage fluids (BALF). Thus DCreg alleviated the T cell mediated airway eosinophilic inflammation in vivo.In summary, we demonstrate the pulmonary fibroblast-like stromal cells can induce immature DCs to differentiate into regulatory DCs which inhibit T cell proliferation both in vitro and in vivo. The DCregs not only induce hypo-responsiveness of T cells but also induce generation of regulatory T cells which may be different form Trl and Th3. There is a natural and functional counterpart of DCreg in lung, and infusion with DCreg generated by co-culturing with MPSC could alleviate T cell mediated airway eosinophilic inflammation in vivo. Therefore, we identified a new subset of DCreg in lung, revealed the influences of lung stromal cells on the development and functions of DCs, further demonstrating the important role of organ microenvironment in the induction of immune regulation or maintaince of immune hemotostasis.