Orchestration of inflammation and adaptive immunity in Borrelia burgdorferi-induced arthritis by neutrophil-activating protein A.

OBJECTIVE: Lyme arthritis (LA) is characterized by infiltration of inflammatory cells, mainly neutrophils (polymorphonuclear cells [PMNs]) and T cells, into the joints. This study was undertaken to evaluate the role of the neutrophil-activating protein A (NapA) of Borrelia burgdorferi in eliciting inflammation and in driving the adaptive immune response. METHODS: Levels of NapA, interferon-γ (IFNγ), interleukin-17 (IL-17), and T cell-attracting chemokines were assessed by enzyme-linked immunosorbent assay in synovial fluid from patients with LA. The profile of T cells recruited into the synovia of patients with LA was defined by fluorescence-activated cell sorting analysis. NapA was intraarticularly injected into rat knees, and the cells recruited in synovia were characterized. The role of NapA in recruiting immune cells was confirmed by chemotaxis assays using a Transwell system. RESULTS: NapA, IFNγ, IL-17, CCL2, CCL20, and CXCL10 accumulated in synovial fluid from patients with LA. Accordingly, T cells obtained from these patients produced IFNγ or IL-17, but notably, some produced both cytokines. NapA promoted neutrophil and T lymphocyte recruitment both in vitro and in vivo. Interestingly, the infiltration of T cells not only resulted from the chemotactic activity of NapA but also relied on the chemokines produced by PMNs exposed to NapA. CONCLUSION: We provide evidence that NapA functions as one of the main bacterial products involved in the pathogenesis of LA. Accordingly, we show that, at very early stages of LA, NapA accumulates and, in turn, orchestrates the recruitment of inflammatory cells into the joint cavity. Thereafter, with the contribution of recruited cells, NapA promotes the infiltration of T cells producing IL-17 and/or IFNγ.

CXCL17 Is Dispensable during Hypervirulent Mycobacterium tuberculosis HN878 Infection in Mice.

CXCL17 is a novel mucosal chemokine that mediates myeloid cell recruitment and bactericidal activity and highly expressed in the respiratory tract. However, its role in tuberculosis (TB) immunopathogenesis or protection remains unknown. In this study, we evaluated the function of CXCL17 in a mouse model of aerosol infection with the clinical W-Beijing lineage Mycobacterium tuberculosis hypervirulent HN878 strain. Our results show that CXCL17 production increases in the lung of M. tuberculosis-infected mice during acute and chronic stages of infection. Moreover, in vitro M. tuberculosis infection of epithelial cells and myeloid cells induces production of CXCL17. In humans, lower serum CXCL17 levels are observed among active pulmonary TB patients when compared with subjects with latent TB infection and healthy controls, suggesting a protective role. However, mice treated with rCXCL17 show similar lung bacterial burden and inflammation compared with control animals, despite an increased lung myeloid cell accumulation. Finally, CXCL17-/- mice are not more susceptible to TB than wild-type animals. These findings suggest that CXCL17 is induced in both murine epithelial and myeloid cells upon M. tuberculosis infection and increased expression during human latent TB infection. However, CXCL17 may have a dispensable role during pulmonary TB.

Acquisition of selectin binding and peripheral homing properties by CD4(+) and CD8(+) T cells.

Different T cell subsets exhibit distinct capacities to migrate into peripheral sites of inflammation, and this may in part reflect differential expression of homing receptors and chemokine receptors. Using an adoptive transfer approach, we examined the ability of functionally distinct subsets of T cells to home to a peripheral inflammatory site. The data directly demonstrate the inability of naive T cells and the ability of effector cells to home to inflamed peritoneum. Furthermore, interleukin (IL)-12 directs the differentiation of either CD4(+) or CD8(+) T cells into effector populations that expresses functional E- and P-selectin ligand and that are preferentially recruited into the inflamed peritoneum compared with T cells differentiated in the presence of IL-4. Recruitment can be blocked by anti-E- and -P-selectin antibodies. The presence of antigen in the peritoneum promotes local proliferation of recruited T cells, and significantly amplifies the Th1 polarization of the lymphocytic infiltrate. Preferential recruitment of Th1 cells into the peritoneum is also seen when cytokine response gene 2 (CRG-2)/interferon gamma-inducible protein 10 (IP-10) is used as the sole inflammatory stimulus. We have also found that P-selectin binds only to antigen-specific T cells in draining lymph nodes after immunization, implying that both antigen- and cytokine-mediated signals are required for expression of functional selectin-ligand.

CXCL17-mediated downregulation of type I collagen via MMP1 and miR-29 in skin fibroblasts possibly contributes to the fibrosis in systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is characterized by excessive deposition of collagen in the skin and internal organs. Recent studies have shown that chemokine (C-X-C motif) ligands (CXCLs) are involved in the pathogenesis of SSc. OBJECTIVE: Our aim was to examine the anti-fibrotic potential of CXCL17, a newly discovered chemokine, in cultured skin fibroblasts and in a bleomycin-induced SSc mouse model. Moreover, we examined serum level of CXCL17 in patients with SSc. METHODS: Type I collagen expression was evaluated in SSc skin and cultured fibroblasts treated with CXCL17 using immunoblotting and quantitative reverse transcription-PCR. Serum CXCL17 levels were determined using enzyme-linked immunosorbent assay in 63 patients with SSc and 17 healthy subjects. A bleomycin-induced SSc mouse model was used to evaluate the effect of CXCL17 on skin fibrosis. RESULTS: CXCL17 reduced the expression of type I collagen in healthy control fibroblasts. CXCL17 also induced matrix metalloproteinase 1 (MMP1) and miR-29 expression in fibroblasts, indicating that CXCL17 regulates type I collagen expression in part via post-transcriptional mechanisms through MMP1 and miR-29. We found that local injection of CXCL17 attenuated bleomycin-induced skin fibrosis in mice. CXCL17 levels in SSc skin were lower than those in healthy controls, in contrast to the high serum CXCL17 levels in patients with SSc. The low expression of CXCL17 in SSc skin possibly affects type I collagen accumulation in this disease. CONCLUSION: Our data indicate that understanding CXCL17 signaling may lead to a better therapeutic approach for SSc.

Kynurenic acid and zaprinast diminished CXCL17-evoked pain-related behaviour and enhanced morphine analgesia in a mouse neuropathic pain model.

BACKGROUND: The G protein-coupled receptor 35 (GPR35), is considered important for nociceptive transmission, as suggested by accumulating evidence. This receptor was discovered in 1998; however, a lack of pharmacological tools prevented a complete understanding of its function and how to exploit it therapeutically. We studied the influence of CXCL17, kynurenic acid and zaprinast on nociceptive transmission in naïve and neuropathic mice. Additionally, we investigated the influence of kynurenic acid and zaprinast on morphine effectiveness in neuropathic pain. METHODS: The chronic constriction injury (CCI) of the sciatic nerve in Swiss mice was performed. The CXCL17, kynurenic acid, zaprinast and morphine were injected intrathecally into naive and CCI-exposed mice at day 14. To evaluate tactile and thermal hypersensitivity, the von Frey and cold plate tests were used, respectively. RESULTS: Our results have shown, for the first time, that administration of CXCL17 in naïve mice induced strong pain-related behaviours, as measured by von Frey and cold plate tests. Moreover, we demonstrated that kynurenic acid and zaprinast diminished CXCL17-evoked pain-related behaviours in both tests. Kynurenic acid and zaprinast reduced thermal and tactile hypersensitivity developed by sciatic nerve injury and strongly enhanced the effectiveness of morphine in neuropathy. CONCLUSIONS: Our study highlights the importance of GPR35 as a receptor involved in neuropathic pain development. Therefore, these results suggest that the modulation of GPR35 could become a potential strategy for the treatment of neuropathic pain.

Resistance to imatinib of bcr/abl p190 lymphoblastic leukemia cells.

Around 20% of patients with acute lymphoblastic leukemia are Philadelphia chromosome positive (Ph-positive acute lymphoblastic leukemia) and express the Bcr/Abl tyrosine kinase. Treatment with the tyrosine kinase inhibitor Imatinib is currently standard for chronic myelogenous leukemia, which is also caused by Bcr/Abl. However, Imatinib has shown limited efficacy for treating Ph-positive acute lymphoblastic leukemia. In our study, we have investigated the effect of Imatinib therapy on murine P190 Bcr/Abl lymphoblastic leukemia cells. Three of four cultures were very sensitive to treatment with 5 mumol/L Imatinib. Significant cell death also initially occurred when the same cultures were treated in the presence of stromal support. However, after 6 days, remaining cells started to proliferate vigorously. The Bcr/Abl tyrosine kinase present in the cells that were now able to multiply in the presence of 5 mumol/L Imatinib was still inhibited by the drug. In concordance with this, the Abl ATP-binding pocket domain of Bcr/Abl in the resistant cells did not contain point mutations which would make the protein Imatinib resistant. The effect of stroma in selecting Imatinib-resistant lymphoblasts did not require direct cell-cell contact. SDF-1alpha could substitute for the presence of stromal cells. Our results show that stroma selects Imatinib-resistant Bcr/Abl P190 lymphoblasts that are less dependent on Bcr/Abl tyrosine kinase activity. Therefore, therapy for Ph-positive acute lymphoblastic leukemia, aimed at interfering with the protective effect of stroma in combination with Imatinib, could be of benefit for the eradication of the leukemic cells.

Controlled release of stromal cell-derived factor-1 alpha in situ increases c-kit+ cell homing to the infarcted heart.

Stromal-derived factor 1alpha (SDF-1alpha) is a key stem cell homing factor that is crucial for mobilization of stem cells from bone marrow to peripheral blood and subsequent engraftment to the tissue of diseased organs. It has been reported that SDF-1alpha is transiently over-expressed in ischemic myocardium. Therefore, there may be a limited time window after acute myocardial infarction (AMI) during which stem cells are recruited to injured myocardium for repair. This study aimed at investigating whether controlled release of SDF-1alpha via a novel conjugated poly(ethylene glycol) (PEG) (PEGylated) fibrin patch at the infarct site would increase the rate of stem cell recruitment and offer potential therapeutic benefits. Recombinant mouse SDF-1alpha was covalently bound to the PEGylated fibrinogen as evidenced by immunoprecipitation and western blotting. The PEGylated fibrinogen, bound with recombinant mouse SDF-1alpha, was mixed with thrombin to form the PEGylated fibrin patch. The release kinetics of SDF-1alpha were detected in vitro using enzyme-linked immunosorbent assay. Using a mouse AMI model produced by a ligature on the left anterior descending coronary artery, a PEGylated fibrin patch bound with SDF-1alpha (100 ng) was placed on the surface of the infarct area of the left ventricle. Infarct size, left ventricular (LV) function, and the percentage of sca-1(+)/c-kit(+) cells within the infarct area were measured at days 7, 14, and 28 after AMI. In vitro results showed that SDF-1alpha was successfully bound to the PEGylated fibrin patch and can be released from the patch constantly for up to 10 days. Two weeks after infarction, the myocardial recruitment of c-kit(+) cells was significantly higher in the group treated with the SDF-1alpha PEGylated fibrin patch (n = 9) than in the AMI control group (n = 10) (p < 0.05; 11.20 +/- 1.71% vs. 4.22 +/- 0.96%, respectively). At day 28 post-AMI, unlike the control group, the group with the SDF-1alpha-releasing patch maintained stable release of SDF-1alpha concurrent with additional stem cell homing. Moreover, LV function was significantly better than in the control group. These data demonstrate that the PEGylated fibrin patch based SDF-1alpha delivery can improve the rate of c-kit(+) cell homing and improve LV function in hearts with postinfarction LV remodeling.

Epiphysiodesis with infusion of stromal cell-derived factor-1 in rabbit growth plates.

BACKGROUND: The mechanism of physeal closure is poorly understood, although both mechanical and biological factors may play a role in the process. In this study, we evaluated the effect of the application of a chemokine stromal cell-derived factor-1 (SDF-1) to rabbit physes in vivo with regard to growth inhibition. METHODS: A continuous infusion system consisting of a fenestrated catheter and an osmotic pump were implanted into the right proximal tibial physis of twenty six-week-old New Zealand White rabbits. Ten of the pumps were loaded with human recombinant SDF-1alpha, and ten were loaded with phosphate-buffered saline solution (sham treatment). The left leg was used as the uninvolved control. The growth of the tibiae was followed radiographically for eight weeks, and histologic analysis was performed for both the SDF-1-treated rabbits and the sham-treated rabbits at two, four, and eight-week time-points. RESULTS: Radiographic evaluation showed a significant growth inhibition in the SDF-1alpha-treated physes (4.5 +/- 3.0 mm; p = 0.007) compared with the sham-treated physes after eight weeks. No difference was noted when the sham-treated leg was compared with the contralateral, control leg (0.2 +/- 2.9 mm; p = 0.465). Histologic evaluation showed marked physeal disorganization, narrowing, and proteoglycan loss and a significant decrease in physeal height (p < 0.0001) for the SDF-1-treated group. Reversible growth slowing was noted in the uninvolved, control leg of the SDF-1-treated group at six weeks, with resolution of the difference by eight weeks. CONCLUSIONS: SDF-1 may be used to induce physeal closure through a targeted infusion system. However, transient systemic effects of SDF-1 may exist and must be evaluated further prior to its clinical use for epiphysiodesis.

Efficacy of systemic administration of SDF-1 in a model of vascular insufficiency: support for an endothelium-dependent mechanism.

OBJECTIVE: Studies have reported that administration of stromal cell-derived factor-1 (SDF-1), the ligand for the G-protein coupled receptor CXCR4, increased collateral blood flow in a mouse model of vascular insufficiency via recruitment of endothelial precursor cells (EPC). The present study investigated the contribution of mature endothelial cells in the actions of SDF-1. METHODS: The regulation of SDF-1 and CXCR4 was examined in the rat cornea cauterization (CC) and aortic ring (AR) model. The functional significance of the SDF-1/CXCR4 pathway was explored in cultured endothelial cells, the AR model, and on collateral blood flow in a rat model of vascular insufficiency. RESULTS: In the present study, the CXCR4 transcript was dramatically upregulated in the rat CC and AR explants, systems containing and lacking bone marrow-derived EPCs, respectively. Addition of AMD3100, a selective CXCR4 antagonist, had no effect on vessel growth in the AR alone, but completely inhibited SDF-1 mediated increases in vascular sprouting. In cultured endothelial cells, SDF-1 alone or in combination with vascular endothelial growth factor (VEGF) significantly enhanced cell survival and migration. Finally, systemic administration of SDF-1 in a rat model of arterial insufficiency enhanced collateral blood flow above vehicle control and equal to that of VEGF after 2 weeks of treatment. CONCLUSION: These studies support activation of the SDF-1/CXCR4 axis as a means to promote blood vessel growth and enhance collateral blood flow, at least in part, via direct effects on vascular endothelial cells.

Improved therapeutic effectiveness by combining recombinant CXC chemokine ligand 10 with Cisplatin in solid tumors.

PURPOSE: CXC chemokine ligand 10 (CXCL10) is a potent inhibitor of angiogenesis. We wonder whether the combination of CXCL10 with cisplatin would improve the therapeutic antitumor efficacy. EXPERIMENT DESIGN: We evaluated the antitumor activity of the combination therapy in the immunocompetent C57BL/6 and BALB/c mice bearing LL/2 Lewis lung cancer and CT26 colon adenocarcinoma, respectively. Mice were treated with either CXCL10 s.c. at 25 mug per kg per day once daily for 30 days, cisplatin cycled twice (5 mg/kg i.p. on days 14 and 21 after the initiation of CXCL10), or both agents together. Tumor volume and survival time were observed. Antiangiogenesis of CXCL10 in vivo were determined by alginate capsule models and CD31 immunohistochemistry. Histologic analysis and assessment of apoptotic cells were also conducted in tumor tissues. RESULTS: CXCL10 + cisplatin reduced tumor growth in LL/2 and CT26 tumor model, respectively, more effectively, although cisplatin or CXCL10 individually resulted in suppression of tumor growth and improved survival time of tumor-bearing mice. CXCL10 successfully inhibited angiogenesis as assessed by alginate model and CD31 (P < 0.05). Histologic analysis of tumors exhibited that CXCL10 in combination with cisplatin led to the increased rate of apoptosis, tumor necrosis, and elevated lymphocyte infiltration. CONCLUSIONS: Our data suggest that the combination of CXCL10, a well-tolerated angiogenesis inhibitor, with cisplatin can enhance the antitumor activity. The present findings may be of importance to the further exploration of the potential application of this combined approach in the treatment of lung and colon carcinoma.

The CXCR4 agonist peptide, CTCE-0021, rapidly mobilizes polymorphonuclear neutrophils and hematopoietic progenitor cells into peripheral blood and synergizes with granulocyte colony-stimulating factor.

OBJECTIVE: Mobilization of hematopoietic stem and progenitor cells (HSPC) by stromal cell-derived factor-1 (SDF-1) has been described; however, sustained adenoviral delivery or N-terminal modification was required for effect and could not be demonstrated with native protein. The aim of this study was to further investigate the SDF-1alpha/CXCR4 axis in HSPC mobilization using CTCE-0021, a cyclized CXCR4 agonist peptide, with comparable bioactivity and improved stability relative to SDF-1alpha. METHODS: Peripheral blood cells and hematopoietic progenitor cells (HPC) were quantitated in mice administered single or multiple doses of CTCE-0021 or SDF-1alpha, or mobilized by granulocyte colony-stimulating factor (G-CSF) in combination with CTCE-0021. Proteases, cytokines, and receptors implicated in HSPC mobilization were evaluated to determine mechanism of action. RESULTS: CTCE-0021 dose-dependently elevated blood neutrophils polymorphonuclear neutrophil [PMN] within 5 minutes that peaked after 1 hour and persisted for 24 hours. PMN mobilization could be maintained by daily dosing. CTCE-0021 mobilized colony-forming unit granulocyte macrophage (CFU-GM), burst-forming unit erythroid (BFU-E), and CFU-granulocyte-erythrocyte-monocyte-megakaryocyte (CFU-GEMM) that peaked within 1 hour after administration, and synergistically enhanced both PMN and HSPC mobilization when combined with G-CSF. Mobilization induced by CTCE-0021 was associated with rapid downregulation of CXCR4 expression on HPC. No appreciable changes in proteases implicated in HPC mobilization were observed. Significantly elevated plasma SDF-1 was detected in mobilized mice, which likely represents CTCE-0021. CONCLUSION: These studies indicate that CTCE-0021 is an efficient and rapid mobilizer of PMN and HPC when used alone and shows synergistic activity when used in combination with G-CSF. The mobilizing effect of this peptide appears to be mediated by downregulation of the CXCR4 receptor on HPC and altered chemokine gradient.

Proteoglycans guide SDF-1-induced migration of hematopoietic progenitor cells.

Stromal cell-derived factor-1 (SDF-1) is a chemoattractant involved in hematopoietic progenitor cell (HPC) trafficking to the bone marrow. We studied the role of bone marrow endothelial proteoglycans (PGs) in SDF-1-mediated migration of HPC using a transwell assay. A subclone of progenitor cell line KG-1 (KG-1v) was used, displaying CXCR4-dependent transmigration. Cell surface PGs on bone marrow endothelial cell line 4LHBMEC did not mediate SDF-1-induced transendothelial migration. In contrast, transwell filters precoated with various glycosaminoglycans (GAGs) enhanced migration toward SDF-1. SDF-1-induced migration was reduced by degradation of heparan sulfate in subendothelial matrix produced by 4LHBMEC. The stimulating effect of GAGs was caused by the formation of a stable haptotactic SDF-1 gradient, as SDF-1 bound to immobilized GAGs and triggered migration. Soluble heparan sulfate enhanced SDF-1-induced migration dose-dependently, suggesting that SDF-1-heparan sulfate complexes optimized SDF-1 presentation. In conclusion, we provide evidence that PGs in the subendothelial matrix establish an SDF-1 gradient guiding migrating HPC into the bone marrow.

Adenovirus-mediated delivery of antiangiogenic genes as an antitumor approach.

Based on the observation that the growth of solid tumors is dependent on the formation of new blood vessels, therapeutic strategies aimed at inhibiting angiogenesis have been proposed. A number of proteins with angiostatic activity have been described, but their development as therapeutic agents has been hampered by difficulties in their production and their poor pharmacokinetics. These limitations may be resolved using a gene therapy approach whereby the genes are delivered and expressed in vivo. Here we compared adenoviral delivery of endostatin, proliferin-related protein (PRP), and interferon-inducible protein 10 (IP10) genes. Recombinant adenoviruses carrying the three angiostatic genes express biologically active gene products as determined in vitro in endothelial cell proliferation and migration assays, and in vivo by inhibition of neoangiogenesis in rat chambers. Eradication of established tumors in vivo, in the murine B16F10 melanoma model in immunocompetent mice, was not achieved by intratumoral injection of the different vectors. However, the combination of intravenous plus intratumoral injections allowed rejection of tumors. Ad-PRP or Ad-IP10 were significantly more efficient than Ad-endostatin, leading to complete tumor rejection and prolonged survival in a high proportion of treated animals. These data support the use of in vivo gene delivery approaches to produce high-circulating and local levels of antiangiogenic agents for the therapy of local and metastatic human tumors.

Changing the chemokine gradient: CINC1 crosses the blood-brain barrier.

Chemokines are a large family of small, inducible, secreted, chemoattractant cytokines that are involved in inflammatory processes. It is well known that systemic and CNS infections cause disruption of the blood-brain barrier (BBB); however, it is not clear how chemokines are involved in this process. We studied the pharmacokinetics of the passage of the chemokine cytokine-induced neutrophil chemoattractant-1 (CINC1) from blood to brain after i.v. bolus injection and its efflux out of the brain after i.c.v. injection. Radiolabeled CINC1 was injected i.v. into mice, and the results were determined by multiple-time regression analysis. Using HPLC, we detected intact CINC1 in brain homogenate and blood after i.v. administration. CINC1 accumulated in the cerebral vasculature but also crossed the BBB completely and rapidly. No saturation of the influx was found, suggesting that either CINC1 crossed the BBB by simple diffusion or the dynamic interactions of binding and internalization precluded the self-inhibition typical of a transport system. Furthermore, there was no efflux system, with CINC1 exiting the brain at the same rate as reabsorption of CSF. The CINC1 injected into blood or CSF did not cause any breakdown of the BBB during the course of the experiments. Thus, the influx of CINC1 may alter the "chemokine gradient" across the BBB and therefore affect inflammatory reactions involving the CNS.

In vivo neutralization of endogenous brain fractalkine increases hippocampal TNFalpha and 8-isoprostane production induced by intracerebroventricular injection of LPS.

Fractalkine is a chemokine widely and constitutively expressed in the brain and, as suggested by in vitro studies, it is involved in brain inflammatory responses. In this study, we have investigated the in vivo anti-inflammatory potential of fractalkine in a model of neuroinflammation induced by intracerebroventricular injection of lipopolysaccharide (LPS) in rats. LPS induces a rapid and acute production of the pro-inflammatory cytokine, TNFalpha, in hippocampus and cerebrospinal fluid (CSF), and an increase of 8-isoprostane levels, a marker of oxidative stress, in hippocampus. Although intracerebroventricular injection of fractalkine has no effect on TNFalpha and 8-isoprostane production, neutralization of endogenous fractalkine within the brain with a specific anti-fractalkine antibody potentiates LPS effects. These data emphasize the involvement of constitutive brain fractalkine in the control of inflammatory reaction in CNS.

The CC chemokines MDC and TARC induce platelet activation via CCR4.

While chemokines have received considerable attention for their role in leukocyte chemotaxis, their effects on platelets have not been well described. We found that two CC chemokine receptor 4 (CCR4) ligands, macrophage-derived chemokine (MDC) and thymus and activation-regulated chemokine (TARC) induce concentration-dependent platelet aggregation and calcium flux. Flow cytometric analysis revealed the expression of CCR4 on platelets and a monoclonal antibody (mAb) to CCR4 inhibited MDC- and TARC-induced platelet aggregation, confirming that this effect is mediated through their common receptor CCR4. MDC fully desensitized TARC-induced calcium mobilization in platelets, while TARC was unable to completely desensitize a subsequent MDC response, which is similar to observations made in Th2 CD4(+) lymphocytes and CCR4-transfected cells. Aspirin (ASA) treatment of platelets allowed reversible primary aggregation but inhibited irreversible complete aggregation, suggesting that MDC- and TARC-induced full platelet aggregation is dependent on cyclooxygenase metabolites of arachidonic acid. MDC and TARC were unable to induce platelet aggregation and platelet secretion in washed human platelets, even though they induced a calcium flux, suggesting that plasma components are required for MDC- and TARC-induced platelet aggregation. Since Th2-type cytokines induce the release of MDC and TARC from cells and the expression of these chemokines is increased in Th2-type inflammation, we hypothesize that MDC and TARC may play a role in platelet activation seen in Th2 diseases, such as asthma and atopic dermatitis.

The use of chemokine receptor agonists in stem cell mobilization.

INTRODUCTION: Pharmacological mobilization has been exploited as a means to obtain hematopoietic stem progenitor cells (HSPCs) for hematopoietic reconstitution. HSPCs mobilized from bone marrow into peripheral blood (PB) are a preferred source of stem cells for transplantation, because they are easily accessible and evidence indicates that they engraft faster after transplantation than HSPCs directly harvested from bone marrow (BM) or umbilical cord blood (UCB). AREAS COVERED: Since chemokine-chemokine receptor axes are involved in retention of HSPCs in the BM microenvironment, chemokine receptor agonists have been proposed as therapeutics to facilitate the mobilization process. These compounds include agonists of the CXCR4 receptor expressed on HSPCs (CTCE-0021 and ATI-2341) or chemokines binding to chemokine receptors expressed on granuclocytes and monocytes (e.g., CXCL2, also known as the growth-related oncogene protein-beta (Gro-ß); CCL3, also known as macrophage inflammatory protein-1α (MIP-1α); or CXCL8, also known as IL-8) could be employed alone or in combination with other mobilizing agents (e.g., G-CSF or Plerixafor (AMD3100)). We discuss the current state of knowledge about chemokine receptor agonists and the rationale for their application in mobilization protocols. EXPERT OPINION: Evidence is accumulating that CXCR4 receptor agonists could be employed alone or with other agents as mobilizing drugs. In particular they may provide an alternative for patients that are poor mobilizers.

CXCL14 deficiency in mice attenuates obesity and inhibits feeding behavior in a novel environment.

BACKGROUND: CXCL14 is a chemoattractant for macrophages and immature dendritic cells. We recently reported that CXCL14-deficient (CXCL14(-/-)) female mice in the mixed background are protected from obesity-induced hyperglycemia and insulin resistance. The decreased macrophage infiltration into visceral adipose tissues and the increased insulin sensitivity of skeletal muscle contributed to these phenotypes. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we performed a comprehensive study for the body weight control of CXCL14(-/-) mice in the C57BL/6 background. We show that both male and female CXCL14(-/-) mice have a 7-11% lower body weight compared to CXCL14(+/-) and CXCL14(+/+) mice in adulthood. This is mainly caused by decreased food intake, and not by increased energy expenditure or locomotor activity. Reduced body weight resulting from the CXCL14 deficiency was more pronounced in double mutant CXCL14(-/-)ob/ob and CXCL14(-/-)A(y) mice. In the case of CXCL14(-/-)A(y) mice, oxygen consumption was increased compared to CXCL14(+/-)A(y) mice, in addition to the reduced food intake. In CXCL14(-/-) mice, fasting-induced up-regulation of Npy and Agrp mRNAs in the hypothalamus was blunted. As intracerebroventricular injection of recombinant CXCL14 did not change the food intake of CXCL14(-/-) mice, CXCL14 could indirectly regulate appetite. Intriguingly, the food intake of CXCL14(-/-) mice was significantly repressed when mice were transferred to a novel environment. CONCLUSIONS/SIGNIFICANCE: We demonstrated that CXCL14 is involved in the body weight control leading to the fully obese phenotype in leptin-deficient or A(y) mutant mice. In addition, we obtained evidence indicating that CXCL14 may play an important role in central nervous system regulation of feeding behavior.

In vivo administration of plasmid DNA encoding recombinant immunotoxin DT390-IP-10 attenuates experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a T-cell-mediated autoimmune demyelinating disease. The expression of chemokine receptor CXCR3 on activated T cells is crucial to direct the migration of effector cells into the inflammatory sites and initiate EAE. In this study we tested the effect of a novel recombinant immunotoxin targeting CXCR3(+) cells for EAE prevention. The immunotoxin construct DT390-IP-10-SRalpha consisted of interferon gamma-inducible protein 10 (IP-10), a ligand of CXCR3, as the targeting moiety, and a truncated diphtheria toxin (DT390) as the toxic moiety. In vitro transfection of DT390-IP-10-SRalpha into NIH3T3 cells resulted in expression of DT390-IP-10 which proved highly toxic to activated T cells. To evaluate the effect of DT390-IP-10-SRalpha on EAE prevention in vivo, cationic liposome-embedded DT390-IP-10-SRalpha was injected into the muscle of hind limbs of C57BL/6 mice immunized by myelin basic protein (MBP). DT390-IP-10-SRalpha-treated mice showed a delayed onset of EAE and milder symptoms compared to the mice treated with empty control plasmid or PBS alone. Immunohistochemical staining detected significantly reduced infiltrating CXCR3(+) cells in the inflammatory lesions of CNS from immunotoxin treated mice as compared to the controls. This study suggests that targeting CXCR3(+) T cells with recombinant immunotoxin could be achieved in vivo to delay and ameliorate murine EAE.