Plt Mice Research Articles

Purple-leaf tea (Camellia sinensis L.) ameliorates high-fat diet induced obesity and metabolic disorder through the modulation of the gut microbiota in mice

BackgroundObesity and its associated diseases have become a major world-wide health problem. Purple-leaf Tea (Camellia sinensis L.) (PLT), that is rich of anthocyanins, has been shown to have preventive effects on obesity and metabolic disorders. The intestinal microbiota has been shown to contribute to inflammation, obesity, and several metabolic disorders. However, whether PLT consumption could prevent obesity and diet-induced metabolic diseases by modulating the gut microbiota, is not clearly understood.MethodsIn this study, six-week-old male C57BL/6 J mice were fed a normal diet (ND) or a high fat diet (HFD) without or with PLT for 10 weeks.ResultsPLT modulated the gut microbiota in mice and alleviated the symptoms of HFD-induced metabolic disorders, such as insulin resistance, adipocyte hypertrophy, and hepatic steatosis. PLT increased the diversity of the microbiota and the ratio of Firmicutes to Bacteroidetes. f_Barnesiellaceae, g_Barnesiella, f_Ruminococcaceae, and f_Lachnospiraceae were discriminating faecal bacterial communities of the PLT mice that differed from the HFD mice.ConclusionsThese data indicate that PLT altered the microbial contents of the gut and prevented microbial dysbiosis in the host, and consequently is involved in the modulation of susceptibility to insulin resistance, hepatic diseases, and obesity that are linked to an HFD.

Relationship of Inflammatory Cytokines From M1-Type Microglia/Macrophages at the Injured Site and Lumbar Enlargement With Neuropathic Pain After Spinal Cord Injury in the CCL21 Knockout (plt) Mouse.

Spinal cord injury (SCI) causes loss of normal sensation and often leads to debilitating neuropathic pain (NeP). Chronic NeP develops at or below the SCI lesion in as many as 80% of patients with SCI and may be induced by modulators of neuronal excitability released from activated microglia and macrophages. In the inflammatory response after SCI, different microglia/macrophage populations that are classically activated (M1 phenotype) or alternatively activated (M2 phenotype) have become of great interest. Chemokines have also recently attracted attention in neuron-microglia communication. CCL21 is a chemokine that activates microglia in the central nervous system (CNS) and is expressed only in neurons with an insult or mechanical injury. In this study using an SCI model in mutant (plt) mice with deficient CCL21 expression, we assessed post-SCI NeP and expression of microglia/macrophages and inflammatory cytokines at the injured site and lumbar enlargement. SCI-induced hypersensitivities to mechanical and thermal stimulation were relieved in plt mice compared with those in wild-type (C57BL/6) mice, although there was no difference in motor function. Immunohistochemistry and flow cytometry analysis showed that the phenotype of microglia/macrophages was M1 type-dominant in both types of mice at the lesion site and lumbar enlargement. A decrease of M1-type microglia/macrophages was seen in plt mice compared with wild-type, while the number of M2-type microglia/macrophages did not differ between these mice. In immunoblot analysis, expression of M1-induced cytokines [tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ)] was decreased in plt mice, while that of M2-induced cytokines interleukin-4 (IL-4, IL-10) did not differ in the two types of mice. The results of this study indicate that suppression of expression of inflammatory cytokines by decreasing the number of M1-type microglia/macrophages at the injured site and lumbar enlargement is associated with provision of an environment for reduction of NeP. These findings may be useful for the design of new therapies to alleviate NeP after SCI.

Abstract 550: Hypercholesterolemia Inhibits LPS-Induced CCR7-Dependent Reverse Transmigration of Arterial Intimal Dendritic Cells.

Dendritic cells (DCs) play a key role in chronic inflammatory diseases such as atherosclerosis. Myeloid cells with features of DCs are abundant in the normal arterial intima of mice, in regions that are predisposed to atherosclerosis. Upon induction of hypercholesterolemia, these intimal DCs rapidly engulf lipids and transform into the initial foam cells of nascent atherosclerotic lesions. The function of intimal DCs in normal mice remains unknown. We observed that systemic stimulation of toll-like receptors (TLRs), specifically TLR4 by LPS and TLR3 by Poly(I:C), induced a rapid reduction in the number of DCs in the normal aortic intima. Absence of TUNNEL staining and use of CD11c-hBcl2 transgenic mice suggested that apoptosis did not account for the loss of intimal DCs. Immuno-gold staining for CD11c coupled with scanning electron microscopy of the aortic surface and ex vivo experiments determined that reverse transmigration (RTM) through the intact endothelial monolayer accounts for LPS-induced reduction of intimal DCs. Intimal DC loss was blocked by pretreatment of wild type mice with pertussis toxin or function-blocking anti-CCL19 antibody, and did not occur in CCR7-/- and Plt (CCL19-/- and CCL21-/-) mice. Assessment of CCR7, CCL19, and CCL21 mRNA expression and reciprocal bone marrow transplantation between CCR7-/- or Plt and wild type mice confirmed that RTM is dependent on CCR7 and CCL19 expression by intimal DCs. Feeding LDLR-/- mice a cholesterol rich diet (CRD) for just one week induced foam cell formation and inhibited LPS-induced RTM; however, CCR7 and CCL19 mRNA induction by LPS was not inhibited. These findings suggest that in the normal mouse aorta, in response to inflammatory stimuli, RTM of RIDCs is dependent on CCR7 signaling; whereas in the setting of hypercholesterolemia, CCR7 signaling is altered in lipid-loaded intimal DCs, which inhibits their RTM. Future experiments will investigate the possibility to overcome this inhibition, so that lipid can be removed from the artery wall by exiting lipid-loaded RIDCs.

Murine Fig4 is dispensable for muscle development but required for muscle function.

BackgroundPhosphatidylinositol phosphates (PIPs) are low-abundance phospholipids that participate in a range of cellular processes, including cell migration and membrane traffic. PIP levels and subcellular distribution are regulated by a series of lipid kinases and phosphatases. In skeletal muscle, PIPs and their enzymatic regulators serve critically important functions exemplified by mutations of the PIP phosphatase MTM1 in myotubular myopathy (MTM), a severe muscle disease characterized by impaired muscle structure and abnormal excitation–contraction coupling. FIG4 functions as a PIP phosphatase that participates in both the synthesis and breakdown of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2). Mutation of FIG4 results in a severe neurodegenerative disorder in mice and a progressive peripheral polyneuropathy in humans. The effect of FIG4 mutation on skeletal muscle has yet to be examined.MethodsHerein we characterize the impact of FIG4 on skeletal muscle development and function using the spontaneously occurring mouse mutant pale tremor (plt), a mouse line with a loss of function mutation in Fig4.ResultsIn plt mice, we characterized abnormalities in skeletal muscle, including reduced muscle size and specific force generation. We also uncovered ultrastructural abnormalities and increased programmed cell death. Conversely, we detected no structural or functional abnormalities to suggest impairment of excitation–contraction coupling, a process previously shown to be influenced by PI(3,5)P2 levels. Conditional rescue of Fig4 mutation in neurons prevented overt muscle weakness and the development of obvious muscle abnormalities, suggesting that the changes observed in the plt mice were primarily related to denervation of skeletal muscle. On the basis of the ability of reduced FIG4 levels to rescue aspects of Mtmr2-dependent neuropathy, we evaluated the effect of Fig4 haploinsufficiency on the myopathy of Mtm1-knockout mice. Male mice with a compound Fig4+/−/Mtm1–/Y genotype displayed no improvements in muscle histology, muscle size or overall survival, indicating that FIG4 reduction does not ameliorate the Mtm1-knockout phenotype.ConclusionsOverall, these data indicate that loss of Fig4 impairs skeletal muscle function but does not significantly affect its structural development.

Demyelination as an Early Pathophysiological Feature in CMT4J (P05.157)

Objective: To delineate segmental demyelination in FIG4 deficiency and its underlying pathogenic mechanism. Background FIG4 is a phosphoinositide phosphatase. FIG4 deficiency causes severe neuronal degeneration and demyelination in patients with recessive Charcot-Marie-Tooth type-4J (CMT4J) and pale tremor mice with Fig4 null. Our recent publication has demonstrated a high level of FIG4 expressed in myelinating Schwann cells throughout development and adulthood, suggesting its important function in myelination (Guo et al, JNEN 2011). However, research in FIG4 has been focused on neuronal degeneration. Demyelination has not been well studied. Design/Methods: Young plt mice and patients with CMT4J at their early stage were examined by electrophysiology, histology and protein chemistry. Results: The mouse sciatic nerves at the age of P21 days were analyzed by teased nerve fiber study. The percentage of segmental demyelination was increased in plt nerves 14.3±1.3% (n=3), compared with 1.9±1.6% in wild=type nerves (n=3, p Conclusions: Demyelination may take place early as a prominant pathological feature in CMT4J and shows a non-uniform pattern imitating acquired demyelination. The demyelination may be related to an up-regulation of Schwann cell dedifferentiation signaling, c-Jun, a novel finding in FIG4 deficiency. Supported by: MDA115087. Disclosure: Dr. Yan has nothing to disclose. Dr. Martyn has nothing to disclose. Dr. Miller has nothing to disclose. Dr. Shy has nothing to disclose. Dr. Li has nothing to disclose.

Distinct pathogenic processes between Fig4-deficient motor and sensory neurons

Loss of function of the FIG4 gene causes Charcot-Marie-Tooth disease (CMT)-4J with many features also found in motor neuron disease (MND). Mechanisms for the degeneration are unknown. We investigated this using Fig4-deficient pale tremor (plt) mice, a mouse model of CMT4J. Ultrastructural studies in sensory neurons of dorsal root ganglion (DRG) confirmed abundant vacuoles with membrane disruption. The vacuoles became detectable as early as postnatal day 4 in the DRG. However, the vacuoles were absent or minimal in the spinal motor neurons or cortical neurons in 2- to 5-week-old plt mice. Instead, a large number of electron-dense organelles, reminiscent of those in lysosomal storage disorders, accumulated in the motor neurons, but not in the sensory neurons of DRG. This accumulation was associated with increased levels of lysosomal proteins, such as LAMP2 and NPC1, but not mannose-6-phosphate receptor, an endosomal protein that is usually excluded from the lysosomes. Our results suggest that Fig4 deficiency affects motor neurons differently from sensory neurons by mechanisms involving excessive retention of molecules in lysosomes or disruption of vacuolated organelles. These two distinct pathological changes may contribute to neuronal degeneration.

Altered antibody production and helper T cell function in mice lacking chemokines CCL19 and CCL21-Ser

The roles of chemokines CCL19 and CCL21 in Ab production were investigated using plt mutant mice, which lack expression of CCL19 and CCL21-ser in their lymphoid organs. In these mice, the Th response has been shown to tend towards the Th1 type because of accumulation of inflammatory dendritic cells. When plt mice were immunized with 100 μg OVA in CFA, the number of Ab-forming cells in the draining LN, and serum concentrations of OVA-specific IgM and IgG Ab, were very close to those of the control, yet IgG2a Ab in plt mice was increased. In vitro IFN-γ production by the draining LN cells of plt mice was increased. In addition, the ability of helper T cells from plt mice to stimulate Ab production in vitro was prolonged. Also, in the plt mice, in vivo challenge with OVA in incomplete Freund's adjuvant elicited a stronger IgG2a response and a weaker IgG1 response, which is suggestive of a Th1-dominant response. Similar findings were obtained when mice were immunized with 100 μg OVA in alum, except that with alum the increases observed in plt mice were IgG1 produced in vivo and IL-4 produced in vitro by draining LN cells. Furthermore, immunization with alum adjuvant also induced a prolonged in vitro recall response of IFN-γ and IL-4. These findings indicate that plt mice mount an anti-OVA Ab response, and suggest that CCL19 and CCL21 induce prompt Ab responses to antigen, and negatively regulate helper T cell responses in vivo.

Knockdown of CCR7 or Its Ligands Causes a Loss of Central Nervous System Involvement in Notch1 Induced T-ALL

Intensive chemotherapy helps achieve remission in the vast majority of acute lymphoblastic leukemia (ALL) patients. A subset of these cases, T-cell ALL (T-ALL), fare significantly worse, presenting with more severe disease, lower remission rates and a greater likelihood of relapse, particularly within the central nervous system (CNS). All modern treatment protocols involve intensive prophylactic CNS chemotherapy and cranial irradiation. The dramatic increase in overall survival is thought to be worth the significant long term side effects associated with CNS treatment such as secondary tumors, neurocognitive deficits and neuroendocrine disorders. Despite its significance, very little is known about the basic mechanisms of leukemic lymphocyte infiltration into the CNS. Over 50% of T-ALL patients have activating mutations in the Notch1 protein. According to our gene expression profiling results, Notch1 activation leads to the induction of CCR7, a chemokine receptor known to play a role in normal lymphocyte homing in extramedullary tissues and CNS. To study CNS infiltration, we generated two murine models expressing activated Notch1 (N1-IC), a bone marrow transplant (WT/WTN1) and a conditional knockin. Both developed an aggressive T-ALL with immature T-cells infiltrating into secondary lymphoid tissues, but more importantly, they had CNS involvement. Numerous malignant appearing CD3 positive cells were found in the leptomeningeal space of the brain in both mouse models. When we transplanted CCR7 knock out bone marrow cells expressing N1-IC, the recipient mice (WT/CCR7KON1) developed an aggressive T-ALL as before. However, a close examination of the brain failed to show any leukemic infiltration in the host mice. In agreement, wild-type N1-IC positive cells transplanted into plt mice (plt/ WTN1) lacking the CCR7 ligands, CCL19 and CCL21, induced T-cell leukemia, but again, these cells failed to infiltrate into the CNS. In addition, WT/CCR7KON1 and plt/WTN1 mice survived longer than WT/WTN1 mice (61 and 71 vs 47 days, p<0.0001). Using real time PCR and FACS, we quantified the levels of CCR7 in 7 human T-ALL cell lines and found that all but one expressed CCR7 at varying levels. The highest expressing, CEM, and non-expressing, DND41, cell lines were transplanted into alymphoid Rag2−/−gc−/− hosts and rapid leukemic infiltration of both lines was monitored by bioluminescent imaging. We found dramatic infiltration of CEM cells, but not DND41 cells into the CNS of the recipients. Moreover, the overexpression of CCR7 in the non-expressing DND41 cells caused them to acquire the ability to infiltrate into the CNS of the hosts. These results strongly implicate CCR7 as a major mediator of CNS involvement in T-cell leukemia and would suggest that targeting the Notch1 or CCR7 pathways could provide an effective CNS therapy with reduced long term morbidity.

Autocrine and Paracrine Chemokine Receptor 7 Activation in Head and Neck Cancer: Implications for Therapy

The chemokine receptor 7 (CCR7) mediates survival and invasiveness of metastatic squamous cell carcinoma of the head and neck (SCCHN) to regional lymph nodes. Constitutive prosurvival signaling by the phosphoinositide-3 kinase/Akt pathway has been observed in SCCHN cells independent of epidermal growth factor receptor (EGFR) signaling. Human SCCHN cell lines were treated with agents that block or activate CCR7-mediated signaling, and Akt activation, cell viability in the presence or absence of EGFR inhibition, and cisplatin-induced apoptosis (in the presence or absence of Akt inhibition) were assessed by immunoblotting, the MTT assay, and the detection of annexin V, respectively. Expression and secretion of chemokines by primary tumors, metastatic nodes, and benign nodes of patients with SCCHN were determined by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The role of paracrine activation of CCR7 on tumor growth was analyzed by comparing the growth of orthotopic tumors derived from B7E3 murine oral carcinoma cells in wild-type BALB/c mice, in paucity of lymphoid T cell (plt, deficient in CCL19 and CCL21 expression) mice, and in plt mice in which the implanted B7E3 cells overexpressed CCR7 (n = 14 mice per group). In the absence of exogenous ligand treatment, blockade of CCR7 signaling reduced levels of phosphorylated (activated) Akt and decreased SCCHN cell viability by up to 59% (95% confidence interval [CI] = 58.2% to 59.8%), enhancing the effect of EGFR inhibition. CCR7 stimulation protected metastatic SCCHN cells from cisplatin-induced apoptosis in an Akt-dependent manner. Metastatic nodes expressed and secreted higher levels of CCL19 than benign nodes or primary tumors. CCR7-positive murine SCCHN tumors grew more slowly in plt mice than in control BALB/c mice (mean average tumor volume on day 20 = 12.2 and 26.5 mm(3), respectively; difference = 14.3 mm(3), 95% CI = 12.3 to 17.1 mm(3)). Secretion of CCL19 and CCL21 by SCCHN cells and by paracrine sources combine to promote activation of CCR7 prosurvival signaling associated with tumor progression and disease relapse. CCR7 and its cognate chemokines may be useful biomarkers of SCCHN progression, and blockade of CCR7-mediated signaling may enhance the efficacy of platinum- and EGFR-based therapies.

Infectious diseases: Too little, too late for tuberculosis

Infectious diseases: Too little, too late for tuberculosis

Regulatory Role of Lymphoid Chemokine CCL19 and CCL21 in the Control of Allergic Rhinitis

The lymphoid chemokines CCL19 and CCL21 are known to be crucial both for lymphoid cell trafficking and for the structural organization of lymphoid tissues such as nasopharynx-associated lymphoid tissue (NALT). However, their role in allergic responses remains unclear, and so our current study aims to shed light on the role of CCL19/CCL21 in the development of allergic rhinitis. After nasal challenge with OVA, OVA-sensitized plt (paucity of lymph node T cells) mice, which are deficient in CCL19/CCL21, showed more severe allergic symptoms than did identically treated wild-type mice. OVA-specific IgE production, eosinophil infiltration, and Th2 responses were enhanced in the upper airway of plt mice. Moreover, in plt mice, the number of CD4(+)CD25(+) regulatory T cells declined in the secondary lymphoid tissues, whereas the number of Th2-inducer-type CD8alpha(-)CD11b(+) myeloid dendritic cells (m-DCs) increased in cervical lymph nodes and NALT. Nasal administration of the plasmid-encoding DNA of CCL19 resulted in the reduction of m-DCs in the secondary lymphoid tissues and the suppression of allergic responses in plt mice. These results suggest that CCL19/CCL21 act as regulatory chemokines for the control of airway allergic disease and so may offer a new strategy for the control of allergic disease.

Role of CCL21 and CCL19 in allergic inflammation in the ovalbumin-specific murine asthmatic model

Dendritic cells are the most powerful of the antigen-presenting cells and are known to play important roles in sensitization and inflammation in allergen-specific asthma. Various cytokines and chemokines are involved in the maturation and activation of dendritic cells. Among them is CC chemokine ligand (CCL)21, a key chemokine in the entry of naive T cells and antigen-stimulated dendritic cells into the T-cell zones of secondary lymphoid organs, which is a critical process in antigen-specific T-cell activation. We studied the role of CCL21 in airway inflammation in asthma by using BALB/c-plt/plt (plt) mice, which possess genetic defects in expression of both CCL21 and CCL19. Plt and control BALB/c mice were immunized with ovalbumin and alum 4 times and thereafter were subjected to a 2-week regimen of ovalbumin inhalation. In plt mice, ovalbumin-specific IgE response was delayed compared with control BALB/c mice, but they had the same level of response after final immunization. Although airway inflammation and response to acetylcholine were significantly reduced compared with BALB/c mice, significant eosinophilic inflammation and hyperresponsiveness were also observed in plt mice after 2 weeks of inhalation. Four weeks after cessation of inhalation, airway inflammation and hyperresponsiveness in plt mice were greater than in BALB/c mice. At the time of resolution of airway inflammation, IL-10 production was enhanced in BALB/c mice but not in plt mice. The chemokines CCL21 and CCL19 were critical for resolution of airway inflammation. The findings about the chemokines for induction and resolution of inflammation are key to establishing a new strategy for asthma immunotherapy.

Permanent Survival of Fully MHC-Mismatched Islet Allografts by Targeting a Single Chemokine Receptor Pathway

Chemokine receptor blockade can diminish the recruitment of host effector cells and prolong allograft survival, but little is known of the role of chemokine receptors in promoting host sensitization. We engrafted fully allogeneic islets into streptozotocin-treated normal mice or mice with the autosomal recessive paucity of lymph node T cell (plt) mutation; the latter lack secondary lymphoid expression of the CCR7 ligands, secondary lymphoid organ chemokine (CCL21) and EBV-induced molecule-1 ligand chemokine (CCL19). plt mice showed permanent survival of islets engrafted under the kidney capsule, whereas controls rejected islet allografts in 12 days (p < 0.001), and consistent with this, plt mice had normal allogeneic T cell responses, but deficient migration of donor dendritic cell to draining lymph nodes. Peritransplant i.v. injection of donor splenocytes caused plt recipients to reject their allografts by 12 days, and sensitization at 60 days posttransplant of plt mice with well-functioning allografts restored acute rejection. Finally, islet allografts transplanted intrahepatically in plt mice were rejected approximately 12 days posttransplant, like controls, as were primarily revascularized cardiac allografts. These data show that the chemokine-directed homing of donor dendritic cell to secondary lymphoid tissues is essential for host sensitization and allograft rejection. Interruption of such homing can prevent T cell priming and islet allograft rejection despite normal T and B cell functions of the recipient, with potential clinical implications.

CXCL9 Antagonism Further Extends Prolonged Cardiac Allograft Survival in CCL19/CCL21‐Deficient Mice

CCL19/MIP-3beta and CCL21/SLC are essential for chemotactic recruitment of mature dendritic cells (DC) to T-cell areas of secondary lymphoid tissue. Paucity of lymph node T-cells (plt/plt) mice lack CCL21-serine (ser) and CCL19 expression. We tested plt/plt and wild type (wt) BALB/c (H2d) mice as recipients of heart or skin allografts from C57BL/10J (H2b) donors. Donor DC trafficking to secondary lymphoid tissue was markedly reduced in plt heart but not skin allograft recipients. Heart, but not skin grafts survived significantly longer in plt recipients. Accordingly, T cells from plt heart transplant recipients demonstrated poor anti-donor responses in ex vivo MLR, compared to wt heart or wt and plt skin recipients. Moreover, donor-reactive T cells from plt heart recipients exhibited Th2-skewing in comparison to T cells from wt heart or skin graft recipients. Anti-CXCL9/Mig was administered for 2 weeks post-transplant to determine whether impairment of activated T-cell migration could further prolong cardiac allograft survival in plt recipients. CXCL9-antagonism extended graft survival significantly only in plt mice, likely due, in part, to retention of alloactivated T cells in secondary lymphoid tissue/reduction of graft-infiltrating T cells. Thus, targeting DC and activated T-cell migration concomitantly has additive effects in prolonging heart graft survival with potential for therapeutic application.

Sphingosine 1-phosphate receptor activation: evidence for distinct chemokine compartments

Introduction: FTY720 is a sphingosine derived immunomodulator that causes T cell egress from peripheral blood and spleen to peripheral lymph nodes through a mechanism dependent on activation of the sphingosine 1-phosphate receptor and sensitization of the CCL19/CCR7 chemokine system. The purposes of this study were to determine if FTY720 enhances T cell migration to other chemokine systems and by what mechanism, and if a chemokine dependent compartmental model of FTY720 enhanced T cell migration exists. Methods: In vivo migration assays were performed with wild type or various knockout (KO) mice strains receiving FTY720 (0.3 mg/kg by gavage); thymus, peripheral blood, spleen, and peripheral lymph nodes were harvested after 18 hours; and T cell distribution was determined by flow cytometry. Control mice received water only. Adoptive transfer assays were performed with CSFE labelled wild type or KO splenocytes injected into wild type or various KO mice strains. All mice were transferred with 2.0 × 107 splenocytes; the experimental group received FTY720, the control group received water. Peripheral blood, spleen, and peripheral lymph nodes were harvested after 18 hours and T cell distribution was determined by flow cytometry. All experiments were performed 3 times with 3 mice per experimental and control groups. Standard deviations and p-values were calculated with Student’s t-test. Results: As shown in Figure 1, in wild-type but not CCR2−/−, CCL2−/−, or plt mice total T cell numbers were reduced in the thymus following FTY720 administration compared to controls. Peripheral blood total T cell numbers were significantly reduced in all mice strains but plt mice had statistically more total T cells then wild type, CCR2−/−, or CCL2−/−. Sequestration of T cells in LN occurred in wild type, plt, and CCL2−/− but not CCR2−/− mice compared to controls; T cell accumulation was significantly greater in wild type compared to plt or CCL2−/− mice. T cell splenic egress was seen in wild type, plt, CCR2−/−, and CCL2−/− strains compared to controls. As shown in Figure 2, CCR2−/− transferred T cells fail to accumulate in the LN in both wild type and plt recipients following FTY720 administration compared to controls and to B6 T cells transferred into the same strains. Conclusions: We conclude that FTY720 enhancement of thymic T cell egress is dependent on the CCR7 and CCR2 chemokine system. LN T cell sequestration is dependent on both CCR2 and CCR7 chemokine systems but also display a CCR7 and CCR2 independent method of T cell accumulation. FTY720 enhance T cell migration occurs in a compartmentalized fashion where the thymus, peripheral blood, peripheral lymph node, and spleen compartments appear to be independent of one another.

The Immune Modulator FTY720 Targets Sphingosine 1-Phosphate Receptors

Immunosuppressant drugs such as cyclosporin have allowed widespread organ transplantation, but their utility remains limited by toxicities, and they are ineffective in chronic management of autoimmune diseases such as multiple sclerosis. In contrast, the immune modulating drug FTY720 is efficacious in a variety of transplant and autoimmune models without inducing a generalized immunosuppressed state and is effective in human kidney transplantation. FTY720 elicits a lymphopenia resulting from a reversible redistribution of lymphocytes from circulation to secondary lymphoid tissues by unknown mechanisms. Using FTY720 and several analogs, we show now that FTY720 is phosphorylated by sphingosine kinase; the phosphorylated compound is a potent agonist at four sphingosine 1-phosphate receptors and represents the therapeutic principle in a rodent model of multiple sclerosis. Our results suggest that FTY720, after phosphorylation, acts through sphingosine 1-phosphate signaling pathways to modulate chemotactic responses and lymphocyte trafficking.

CC chemokine receptor 7-dependent and -independent pathways for lymphocyte homing: modulation by FTY720.

Cognate interaction of chemokine receptor CCR7 on lymphocytes with its ligands CCL19 and CCL21 expressed on high endothelial venules (HEVs) is essential for effective migration of T and B cells across HEVs into secondary lymphoid organs. Plt mice, which lack expression of CCL19 and CCL21-ser, both ligands for CCR7 on HEVs, as well as CCR7-deficient mice, have a defective cell migration and reduced homing of lymphocytes. FTY720, a novel immunosuppressant, causes a reduction of lymphocytes in peripheral blood and tissues and their sequestration into lymphoid tissues. In this study we demonstrate that FTY720 rescues the homing defect in both CCR7(-/-) mice and plt mice. After FTY720 treatment, the number of CD4(+) and CD8(+) T cells as well as B cells in peripheral blood is reduced while pertussis toxin-sensitive homing into peripheral lymph nodes, mesenteric lymph node, and Peyer's patches is increased. Immunohistology demonstrates that FTY720 enables these cells to enter lymphoid tissue through HEVs. Thus, our data suggest an alternative G-alpha(i)-dependent, CCR7-CCL19/CCL21-independent mechanism for lymphocyte homing through HEVs which is strongly augmented in the presence of FTY720.

Mice lacking expression of the chemokines CCL21-ser and CCL19 (plt mice) demonstrate delayed but enhanced T cell immune responses.

The paucity of lymph node T cells (plt) mutation leads to a loss of CCL21 and CCL19 expression in secondary lymphoid organs. plt mice have defects in the migration of naive T cells and activated dendritic cells into the T cell zones of lymphoid organs, suggesting that they would have defects in T cell immune responses. We now demonstrate T cell responses in plt mice are delayed but ultimately enhanced. Responses to contact sensitization are decreased at day 2 after priming but increased at day 6. After subcutaneous immunization, antigen-specific T cell proliferation and cytokine production in plt mice are increased and remain markedly elevated for at least 8 wk. Compared with wild-type mice, a proportion of T cell response in plt mice are shifted to the spleen, and prior splenectomy reduces the T cell response in draining lymph nodes. After immunization of plt mice, T cells and dendritic cells colocalize in the superficial cortex of lymph nodes and in splenic bridging channels, but not in T cell zones. These results demonstrate that plt mice mount robust T cell responses despite the failure of naive T cells and activated dendritic cells to enter the thymus dependent areas of secondary lymphoid organs.

Gene duplications at the chemokine locus on mouse chromosome 4: multiple strain-specific haplotypes and the deletion of secondary lymphoid-organ chemokine and EBI-1 ligand chemokine genes in the plt mutation.

The murine paucity of lymph node T cell (plt) mutation leads to abnormalities in leukocyte migration and immune response. The causative defect is thought to be a loss of secondary lymphoid-organ chemokine (SLC) expression in lymphoid tissues. We now find that the plt defect is due to the loss of both SLC and EBI-1 ligand chemokine (ELC) expression in secondary lymphoid organs. In an examination of the plt locus, we find that commonly used inbred mouse strains demonstrate at least three different haplotypes. Polymorphism at this locus is due to duplications of at least four genes, three of them encoding chemokines. At least two cutaneous T cell-attracting chemokine (CTACK), three SLC, and four ELC genes or pseudogenes are present in some haplotypes. All haplotypes share a duplication that includes two SLC genes, which demonstrate different expression patterns, a single functional ELC gene, and an ELC pseudogene. The plt mutation represents a deletion that includes the SLC gene expressed in secondary lymphoid organs and the single functional ELC gene, leaving only an SLC gene that is expressed in lymphatic endothelium and an ELC pseudogene. This lack of CCR7 ligands in the secondary lymphoid organs of plt mice provides a basis for their severe abnormalities in leukocyte migration and immune response.

The reduced expression of 6Ckine in the plt mouse results from the deletion of one of two 6Ckine genes.

6Ckine is an unusual chemokine capable of attracting naive T lymphocytes in vitro. It has been recently reported that lack of 6Ckine expression in lymphoid organs is a prominent characteristic of mice homozygous for the paucity of lymph node T cell (plt) mutation. These mice show reduced numbers of T cells in lymph nodes, Peyer's patches, and the white pulp of the spleen. The genetic reason for the lack of 6Ckine expression in the plt mouse, however, has remained unknown. Here we demonstrate that mouse 6Ckine is encoded by two genes, one of which is expressed in lymphoid organs and is deleted in plt mice. A second 6Ckine gene is intact and expressed in the plt mouse.