Extracellular vesicles from dHL-60 cells as delivery vehicles for diverse therapeutics.

Extracellular vesicles (EVs) are membrane-derived heterogeneous vesicles that mediate intercellular communications. They have recently been considered as ideal vehicles for drug-delivery systems, and immune cells are suggested as a potential source for drug-loaded EVs. In this study, we investigated the possibility of neutrophils as a source for drug-loaded EVs. Neutrophil-like differentiated human promyelocytic leukemia cells (dHL-60) produced massive amounts of EVs within 1 h. The dHL-60 cells are also easily loaded with various cargoes such as antibiotics (penicillin), anticancer drug (paclitaxel), chemoattractant (MCP-1), miRNA, and Cas9. The EVs derived from the dHL-60 cells showed efficient incorporation of these cargoes and significant effector functions, such as bactericidal activity, monocyte chemotaxis, and macrophage polarization. Our results suggest that neutrophils or neutrophil-like promyelocytic cells could be an attractive source for drug-delivery EVs.

Effects of adipokine administration to the hypothalamic preoptic area on body temperature in rats.

The effects of adipokine administration to the hypothalamic preoptic area (POA), which is one of the body temperature (BT) regulation centers in the central nervous system, on BT were investigated in male Wistar rats. BT was measured in conscious rats using telemetry. Insulin-like growth factor-1 (IGF-1), interleukin-1ß (IL-1ß), monocyte chemoattractant protein-1 and lipocalin-2 produced hyperthermia, and the effects induced by IL-1ß (25 ng) and IGF-1 (5 µg) were sustainable and remarkable. IL-6 did not show any significant effect. The IGF-1-induced effect was inhibited by pretreatment with IGF binding protein 3 (IGFBP3) or NVP-AEW541 (NVP, a selective inhibitor of type 1 IGF receptor tyrosine kinase, IGF1R TK). NVP-induced inhibition was observed only in the early phase of IGF-1-induced hyperthermia. In addition, IGF-1 increased the IL-1ß concentration in the microdialysate of POA perfusion, but did not increase the IL-1ß concentration in the plasma or the PGE2 concentration in the microdialysate. These findings suggested that IGF-1 produced hyperthermia, which was mediated, at least a part, through an increased IL-1ß concentration after activation of IGF1R TK in the POA, and the IGF-IGFBP system possibly participates in BT homeostasis in the POA.

Chemokine CCL2 impairs spatial memory and cognition in rats via influencing inflammation, glutamate metabolism and apoptosis-associated genes expression- a potential mechanism for HIV-associated neurocognitive disorder.

AIMS: To explore the role of chemokine CC motif ligand 2 (CCL2) in spatial memory and cognition impairment, and the underlying mechanisms focused on inflammatory, glutamate metabolistic and apoptotic- associated mRNA expression. MATERIALS AND METHODS: Stereotaxic surgery was performed here to establish a rat model by bilateral intra-hippocampal injection of CCL2. Morris water maze (MWM) and Novel object recognition test (NORT) were used to assess the learning, memory and cognitive ability respectively. RT-PCR was used to detect the relative mRNA expression of inflammatory, glutamate metabolistic and apoptotic- associated indexes. Nissl and TUNEL staining were performed to observe the morphological changes of hippocampal CA1 zone and quantified the apoptosis of hippocampal neurons of CA1 zones respectively. KEY FINDINGS: We found CCL2 injured cognitive function in rats. Six days after CCL2 injection, we revealed the following obvious mRNA expression changes: (1) increasing of the neuroinflammatory cytokines IL-1ß, CXCL-10, IL-6; (2) decreasing of the glutamate transporters GLT-1 and GLAST and increasing of PAG; (3) increasing of the apoptotic genes caspase-8, caspase-3 and Bax, while decreasing the anti-apoptotic gene Bcl-2. Further, Nissl staining and TUNEL confirmed the injury of the structure of hippocampal CA1 zones and the apoptosis of hippocampal neurons. SIGNIFICANCE: Our results indicated that CCL2 impaired spatial memory and cognition, the involving mechanisms may link to the up-regulation of mRNA expression of the three major pathological events: inflammation, excitotoxicity and neuronal apoptosis, which were involved in HIV-associated neurocognitive disorder (HAND). Taken together, these findings suggest a potential therapeutic strategy against CCL2.

CCL2-Mediated Reversal of Impaired Skin Wound Healing in Diabetic Mice by Normalization of Neovascularization and Collagen Accumulation.

Patients with diabetes frequently present with complications such as impaired skin wound healing. Skin wound sites display a markedly enhanced expression of CCL2, a potent macrophage chemoattractant, together with macrophage infiltration during the early inflammatory phase in skin wound healing of healthy individuals, but the association of CCL2 with delayed skin wound healing in patients with diabetes remains elusive. In this study, we showed that, compared with control mice, mice with streptozotocin-induced diabetes displayed impaired healing after excisional skin injury, with decreased neovascularization, CCL2 expression, and macrophage infiltration. Compromised skin wound healing in mice with diabetes was reversed by the administration of topical CCL2 immediately after the injury, as evidenced by normalization of wound closure rates, neovascularization, collagen accumulation, and infiltration of macrophages expressing vascular endothelial growth factor, a potent angiogenic factor, and transforming growth factor-ß. CCL2 treatment further increased the accumulation of endothelial progenitor cells at the wound sites of mice with diabetes and eventually accelerated neovascularization. Thus, the topical application of CCL2 can be an effective therapeutic option for the treatment of patients with diabetes with defective wound repair, promoting neovascularization and collagen accumulation at skin wound sites.

Distinct release strategies are required to modulate macrophage phenotype in young versus aged animals.

The role of innate immunity and macrophages in the host response to biomaterials has received renewed attention. A context-dependent spectrum of macrophage phenotypes are shown to affect tissue integration and performance of implanted biomaterials and medical devices. Recent studies by our group demonstrated that the host response in aged animals was characterized by delayed macrophage recruitment, differences in marker expression and a shifted pro-inflammatory (M1) response, associated with an unresolved host response in the long-term. The present work sought to study the effects of single and sequential cytokine delivery regimens in aged mice to restore delayed recruitment of macrophages and shift the inflammatory host response towards an M2-like phenotype, using MCP-1 (macrophage chemotactic protein-1) and IL-4 (interleukin-4), respectively. Implantation of cytokine-eluting implants showed a preserved response to MCP-1 in both young and aged animals, restoring delayed macrophage recruitment in aged mice. However, the response elicited by IL-4, sequential delivery of MCP-1/IL-4 and coating components was distinct in young versus aged mice. While single delivery of IL-4 did not counteract the high inflammatory response observed in aged mice, the sequential delivery of MCP-1/IL-4 was capable of restoring both recruitment and shifting the macrophage response towards an M2-like phenotype, associated with decreased implant scarring in the long-term. In young mice, sequential delivery was not as effective as IL-4 alone at promoting an M2-like response, but did result in a reduction of M1 macrophages and capsule deposition downstream. These results demonstrate that a proper understanding of patient/context-dependent biological responses are needed to design biomaterial-based therapies with improved outcomes in the setting of aging.

Co-delivery of RNAi and chemokine by polyarginine nanocapsules enables the modulation of myeloid-derived suppressor cells.

Myeloid-Derived Suppressor Cells (MDSCs), immunosuppressive cells that promote tumor growth, represent an attractive target in cancer immunotherapy. However, the clinical success of this strategy is limited by the lack of efficient drug delivery vehicles targeting this cell compartment. The objective of this work was to develop a delivery carrier, multilayer polymer nanocapsules, with the capacity to co-encapsulate two types of immunomodulatory drugs, a chemokine and an RNAi sequence, aimed at reverting MDSC-mediated immunosuppression. The chemokine CCL2, intended to attract monocyte-macrophage MDSCs, was encapsulated within the L2 inverse micellar aqueous domains of the lipid core of these nanocapsules. On the other hand, two different RNAi sequences that modulate the CCAAT/enhancer-binding protein beta (C/EBPß) pathway, shC/EBPß and miR 142-3p, were successfully associated to their polymer shell. These RNAi sequences were covered by subsequent layers of polyarginine and hyaluronic acid, thereby creating multi-layered assemblies that protected them and facilitated their targeted delivery. The in vitro studies performed in primary MDSCs cultures showed the capacity of miR 142-3p-loaded nanocapsules to reduce the highly immunosuppressive monocyte-macrophage subset. Additionally, the encapsulation of CCL2 within the nanocapsules induced a potent monocyte-macrophage chemoattraction that could be used to direct the therapy to these cell subsets. Finally, in vitro and in vivo studies showed the capacity of shC/EBPß-loaded nanocapsules to downregulate C/EBPß levels in MDSCs and to reduce monocyte differentiation into tumor-associated macrophages in an MCA-203 fibrosarcoma mice model. In conclusion, the multilayer polymer nanocapsules described here are efficient vehicles for the co-delivery of proteins and RNA, and are potential candidates as nanomedicines for the modulation of MDSCs.

Mesenchymal stem cells encapsulated into biomimetic hydrogel scaffold gradually release CCL2 chemokine in situ preserving cytoarchitecture and promoting functional recovery in spinal cord injury.

Spinal cord injury (SCI) is an acute neurodegenerative disorder caused by traumatic damage of the spinal cord. The neuropathological evolution of the primary trauma involves multifactorial processes that exacerbate the pathology, worsening the neurodegeneration and limiting neuroregeneration. This complexity suggests that multi-therapeutic approaches, rather than any single treatment, might be more effective. Encouraging preclinical results indicate that stem cell-based treatments may improve the disease outcome due to their multi-therapeutic ability. Mesenchymal Stem Cells (MSCs) are currently considered one of the most promising approaches. Significant improvement in the behavioral outcome after MSC treatment sustained by hydrogel has been demonstrated. However, it is still not known how hydrogel contribute to the delivery of factors secreted from MSCs and what factors are released in situ. Among different mediators secreted by MSCs after seeding into hydrogel, we have found CCL2 chemokine, which could account for the neuroprotective mechanisms of these cells. CCL2 secreted from human MSCs is delivered efficaciously in the lesioned spinal cord acting not only on recruitment of macrophages, but driving also their conversion to an M2 neuroprotective phenotype. Surprisingly, human CCL2 delivered also plays a key role in preventing motor neuron degeneration in vitro and after spinal cord trauma in vivo, with a significant improvement of the motor performance of the rodent SCI models.

Temporal cascade of inflammatory cytokines and cell-type populations in monocyte chemotactic protein-1 (MCP-1)-mediated aneurysm healing.

BACKGROUND: We have previously shown that monocyte chemotactic protein-1 (MCP-1) promotes aneurysm healing. OBJECTIVE: To determine the temporal cascade and durability of aneurysm healing. METHODS: Murine carotid aneurysms were treated with MCP-1-releasing or poly(lactic-co-glycolic) acid (PLGA)-only coils. Aneurysm healing was assessed by quantitative measurements of intraluminal tissue ingrowth on 5 µm sections by blinded observers. RESULTS: Aneurysm healing occurred in stages characteristic of normal wound healing. The 1st stage (day 3) was characterized by a spike in neutrophils and T cells. The 2nd stage (week 1) was characterized by an influx of macrophages and CD45+ cells significantly greater with MCP-1 than with PLGA (p<0.05). The third stage (week 2-3) was characterized by proliferation of smooth muscle cells and fibroblasts (greater with MCP-1 than with PLGA, p<0.05). The fourth stage (3-6 months) was characterized by leveling off of smooth muscle cells and fibroblasts. M1 macrophages were greater at week 1, whereas M2 macrophages were greater at weeks 2 and 3 with MCP-1 than with PLGA. Interleukin 6 was present early and increased through week 2 (p<0.05 compared with PLGA) then decreased and leveled off through 6 months. Tumour necrosis factor α was present early and remained constant through 6 months. MCP-1 and PLGA treatment had similar rates of tissue ingrowth at early time points, but MCP-1 had a significantly greater tissue ingrowth at week 3 (p<0.05), which persisted for 6 months. CONCLUSIONS: The sequential cascade is consistent with an inflammatory model of injury, repair, and remodeling.

DNA damage mediates changes in neuronal sensitivity induced by the inflammatory mediators, MCP-1 and LPS, and can be reversed by enhancing the DNA repair function of APE1.

Although inflammation-induced peripheral sensitization oftentimes resolves as an injury heals, this sensitization can be pathologically maintained and contribute to chronic inflammatory pain. Numerous inflammatory mediators increase the production of reactive oxygen (ROS) and nitrogen species (RNS) during inflammation and in animal models of chronic neuropathic pain. Our previous studies demonstrate that ROS/RNS and subsequent DNA damage mediate changes in neuronal sensitivity induced by anticancer drugs and by ionizing radiation in sensory neurons, thus we investigated whether inflammation and inflammatory mediators also could cause DNA damage in sensory neurons and whether that DNA damage alters neuronal sensitivity. DNA damage was assessed by pH2A.X expression and the release of the neuropeptide, calcitonin gene-related peptide (CGRP), was measured as an index of neuronal sensitivity. Peripheral inflammation or exposure of cultured sensory neurons to the inflammatory mediators, LPS and MCP-1, elicited DNA damage. Moreover, exposure of sensory neuronal cultures to LPS or MCP-1 resulted in changes in the stimulated release of CGRP, without altering resting release or CGRP content. Genetically enhancing the expression of the DNA repair enzyme, apurinic/apyrimidinic endonuclease (APE1) or treatment with a small-molecule modulator of APE1 DNA repair activity, both which enhance DNA repair, attenuated DNA damage and the changes in neuronal sensitivity elicited by LPS or MCP-1. In conclusion, our studies demonstrate that inflammation or exposure to inflammatory mediators elicits DNA damage in sensory neurons. By enhancing DNA repair, we demonstrate that this DNA damage mediates the alteration of neuronal function induced by inflammatory mediators in peptidergic sensory neurons.

Monocyte Chemotactic Protein-1-Interleukin-6-Osteopontin Pathway of Intra-Aneurysmal Tissue Healing.

BACKGROUND AND PURPOSE: We have previously demonstrated that the local delivery of monocyte chemotactic protein-1 (MCP-1) via an MCP-1-releasing poly(lactic-co-glycolic acid)-coated coil promotes intra-aneurysmal tissue healing. In this study, we demonstrate that interleukin-6 (IL-6) and osteopontin are downstream mediators in the MCP-1-mediated aneurysm-healing pathway. METHODS: Murine carotid aneurysms were created in C57BL/6 mice. Drug-releasing coils (MCP-1, IL-6, and osteopontin) and control poly(lactic-co-glycolic acid) coils were created and then implanted into the aneurysms to evaluate their intra-aneurismal-healing capacity. To investigate the downstream mediators for aneurysm healing, blocking antibodies for IL-6 receptor and osteopontin were given to the mice implanted with the MCP-1-releasing coils. A histological analysis of both murine and human aneurysms was utilized to cross-validate the data. RESULTS: We observed increased expression of IL-6 in MCP-1-coil-treated aneurysms and not in control-poly(lactic-co-glycolic acid)-only-treated aneurysms. MCP-1-mediated intra-aneurysmal healing is inhibited in mice given blocking antibody to IL-6 receptor. MCP-1-mediated intra-aneurysmal healing is also inhibited by blocking antibody to osteopontin. The role of IL-6 in intra-aneurysmal healing is in recruiting of endothelial cells and fibroblasts. Local delivery of osteopontin to murine carotid aneurysms via osteopontin-releasing coil significantly promotes intra-aneurysmal healing, but IL-6-releasing coil does not, suggesting that IL-6 cannot promote aneurysm healing independent of MCP-1. In the MCP-1-mediated aneurysm healing, osteopontin expression is dependent on IL-6; inhibition of IL-6 receptor significantly inhibits osteopontin expression in MCP-1-mediated aneurysm healing. CONCLUSIONS: Our findings suggest that IL-6 and osteopontin are key downstream mediators of MCP-1-mediated intra-aneurysmal healing.

DJ-1 deficiency attenuates expansion of liver progenitor cells through modulating the inflammatory and fibrogenic niches.

Our previous study suggested that DJ-1 has a critical role in initiating an inflammatory response, but its role in the liver progenitor cell (LPC) expansion, a process highly dependent on the inflammatory niche, remains elusive. The objective of this study is to determine the role of DJ-1 in LPC expansion. The correlation of DJ-1 expression with LPC markers was examined in the liver of patients with hepatitis B or hepatitis C virus (HBV and HCV, respectively) infection, primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), nonalcoholic fatty liver disease (NAFLD), cirrhosis or hepatocellular carcinoma (HCC), respectively. The role of DJ-1 in LPC expansion and the formation of LPC-associated fibrosis and inflammation was examined in a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet-induced liver injury murine model. We also determined the ability of hepatic stellate cells (HSCs) in recruiting macrophages in DJ-1 knockout (KO) mice. The expression levels of DJ-1 were upregulated in the liver of HBV, HCV, PBC and PSC patients and DDC-fed mice. Additionally, DJ-1 expression was positively correlated with LPC proliferation in patients with liver injury and mice with DDC exposure. DJ-1 has no direct effect on LPC proliferation. Reduced activation of HSCs and collagen deposition were observed in DJ-1 KO mice. Furthermore, infiltrated CD11b(+)Gr-1(low) macrophages and pro-inflammatory factors (IL-6, TNF-α) were attenuated in DJ-1 KO mice. Mechanistically, we found that HSCs isolated from DJ-1 KO mice had decreased secretion of macrophage-mobilizing chemokines, such as CCL2 and CX3CL1, resulting in impaired macrophage infiltration. DJ-1 positively correlates with LPC expansion during liver injury. DJ-1 deficiency negatively regulates LPC proliferation by impairing the formation of LPC-associated fibrosis and inflammatory niches.

The therapeutic effect of monocyte chemoattractant protein-1 delivered by an electrospun scaffold for hyperglycemia and nephrotic disorders.

Here, we investigated in diabetic mice the therapeutic effect of monocyte chemoattractant protein-1 (MCP-1), locally delivered by an electrospun scaffold, on transplanted islets. This therapeutic scheme is expected to exert a synergistic effect to ameliorate hyperglycemia and its associated nephrotic disorders. The cumulative amount of MCP-1 released from the scaffold in vitro within a 3-week window was 267.77 ± 32.18 ng, without a compromise in bioactivity. After 8 weeks following the transplantation, the islet population stimulated by MCP-1 was 35.14%± 7.23% larger than the non-stimulated islet population. Moreover, MCP-1 increased concentrations of blood insulin and C-peptide 2 by 49.83%± 5.29% and 43.49%± 9.21%, respectively. Consequently, the blood glucose concentration in the MCP-1 group was significantly lower than that in the control group at week 2 post-surgery. MCP-1 also enhanced the tolerance of sudden oral glucose challenge. The rapid decrease of blood creatinine, urine creatinine, and blood urea nitrogen suggested that the recovery of renal functions compromised by hyperglycemia could also be attributed to MCP-1. Our study shed new light on a synergistic strategy to alleviate hyperglycemia and nephrotic disorders in diabetic patients.

Intracerebral administration of recombinant rabies virus expressing GM-CSF prevents the development of rabies after infection with street virus.

Recently it was found that prior immunization with recombinant rabies virus (RABV) expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) (LBNSE-GM-CSF) resulted in high innate/adaptive immune responses and protection against challenge with virulent RABV (Wen et al., JVI, 2011). In this study, the ability of LBNSE-GM-CSF to prevent animals from developing rabies was investigated in mice after infection with lethal doses of street RABV. It was found that intracerebral administration of LBNSE-GM-CSF protected more mice from developing rabies than sham-treated mice as late as day 5 after infection with street RABV. Intracerebral administration of LBNSE-GM-CSF resulted in significantly higher levels of chemokine/cytokine expression and more infiltration of inflammatory and immune cells into the central nervous system (CNS) than sham-administration or administration with UV-inactivated LBNSE-GM-CSF. Enhancement of blood-brain barrier (BBB) permeability and increases in virus neutralizing antibodies (VNA) were also observed in mice treated with LBNSE-GM-CSF. On the other hand, intracerebral administration with UV-inactivated LBNSE-GM-CSF did not increase protection despite the fact that VNA were induced in the periphery. However, intracerebral administration with chemoattractant protein-1 (MCP-1, also termed CCL2) increased significantly the protective efficacy of UV-inactivated LBNSE-GM-CSF. Together these studies confirm that direct administration of LBNSE-GM-CSF can enhance the innate and adaptive immunity as well as the BBB permeability, thus allowing infiltration of inflammatory cells and other immune effectors enter into the CNS to clear the virus and prevent the development of rabies.

Prevention of intrahepatic metastasis of liver cancer by suicide gene therapy and chemokine ligand 2/monocyte chemoattractant protein-1 delivery in mice.

BACKGROUND: The prognosis of patients with hepatocellular carcinoma (HCC) remains poor, largely as a result of intrahepatic metastasis. Using a mouse model of intrahepatic metastasis, we investigated whether chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2/MCP-1) could potentiate the antitumor effects of the herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system. METHODS: Mouse hepatoma cells infected with recombinant adenovirus vectors expressing HSV-tk, CCL2/MCP-1 and LacZ at multiplicities of infection of Ad-tk/Ad-MCP1 = 3/0.03 (T/M(Low)), 3/3 (T/M(High)) and Ad-tk/Ad-LacZ = 3/3 (T/L) were injected into BALB/c mice. RESULTS: Intrahepatic tumor growth was significantly lower in T/M(Low) mice. By contrast, no tumor suppression was observed in T/M(High) mice. The tumor-specific cytolytic activities of splenocytes from T/M(Low) and T/M(High) mice were comparable. Immunohistochemical analysis of liver tissues showed similar infiltration by Mac-1(+) and T cells in these animals, whereas the proportions of classical activated (M1) monocytes/macrophages were significantly higher in T/M(Low) mice. In addition, interleukin-12 production was elevated in these tissues. Vascular endothelial growth factor-A expression and CD31(+) microvessels were increased in T/M(High) mice. CONCLUSIONS: Collectively, these results demonstrate that an adequate amount of CCL2/MCP-1, together with the HSV-tk/GCV system, may induce T helper 1-polarized antitumor effects without inducing tumor angiogenesis in the microenvironment of intrahepatic HCC progression.

An increase in the circulating concentration of monocyte chemoattractant protein-1 elicits systemic insulin resistance irrespective of adipose tissue inflammation in mice.

Chronic inflammation in adipose tissue is thought to be important for the development of insulin resistance in obesity. Furthermore, the level of monocyte chemoattractant protein-1 (MCP-1) is increased not only in adipose tissue but also in the circulation in association with obesity. However, it has remained unclear to what extent the increased circulating level of MCP-1 contributes to insulin resistance. We have now examined the relevance of circulating MCP-1 to the development of insulin resistance in mice. The plasma concentration of MCP-1 was increased chronically or acutely in mice to the level observed in obese animals by chronic subcutaneous infusion of recombinant MCP-1 with an osmotic pump or by acute intravenous infusion of MCP-1 with an infusion pump, respectively. Whole-body metabolic parameters as well as inflammatory changes in adipose tissue were examined. A chronic increase in the circulating level of MCP-1 induced insulin resistance, macrophage infiltration into adipose tissue, and an increase in hepatic triacylglycerol content. An acute increase in the circulating MCP-1 concentration also induced insulin resistance but not macrophage infiltration into adipose tissue. In addition, inhibition of signaling by MCP-1 and its receptor CCR2 by administration of a novel CCR2 antagonist ameliorated insulin resistance in mice fed a high-fat diet without affecting macrophage infiltration into adipose tissue. These data indicate that an increase in the concentration of MCP-1 in the circulation is sufficient to induce systemic insulin resistance irrespective of adipose tissue inflammation.

Dual delivery of VEGF and MCP-1 to support endothelial cell transplantation for therapeutic vascularization.

Transplantation of endothelial cells (EC) for therapeutic vascularization is a promising approach in tissue engineering but has yet to be proven effective in clinical trials. This cell-based therapy is hindered by significant apoptosis of EC upon transplantation as well as poor recruitment of host mural cells to stabilize nascent vessels. Here, we address these deficiencies by augmenting endothelial cell transplantation with dual delivery of vascular endothelial growth factor (VEGF) - to improve survival of transplanted EC - and monocyte chemotactic protein-1 (MCP-1) - to induce mural cell recruitment. We produced alginate microparticles that deliver VEGF and MCP-1 with distinct release kinetics and that can be integrated into a collagen/fibronectin (protein) gel construct for delivery of EC. Combined delivery of VEGF and MCP-1 increased functional vessel formation from transplanted EC and also led to a higher number of smooth muscle cell-invested vessels than did EC therapy alone. Despite the well-known role of MCP-1 in inflammation, these beneficial effects were accomplished without a long-term increase in monocyte/macrophage recruitment or a shift to a pro-inflammatory (M1) macrophage phenotype. Overall, these data suggest a potential benefit of combined delivery of MCP-1 and VEGF from EC-containing hydrogels as a strategy for therapeutic vascularization.

Selective inhibition of CCR2 expressing lymphomyeloid cells in experimental autoimmune encephalomyelitis by a GM-CSF-MCP1 fusokine.

We describe the generation of a fusion cytokine consisting of GM-CSF in tandem with N-terminal-truncated MCP-1 (6-76), hereafter GMME1. Treatment of activated T cells with recombinant GMME1 protein leads to proinflammatory cytokine reduction and apoptosis via a CCR2-restricted pathway. Similarly, cell death is triggered in macrophages cultured with GMME1, while an inhibition of Ab production from plasma cells is observed. Treatment of CD4 T cells derived from experimental autoimmune encephalomyelitis mice with GMME1 leads to p38 hyperphosphorylation, inhibition of p44/42, AKT and STAT3 phosphorylation, and caspase-3 activation. GMME1 administration to experimental autoimmune encephalomyelitis mice suppresses symptomatic disease and correlates with decreased levels of inflammatory cytokines including IL-17, MOG-specific Ab titers, and blockade of CD4 and CD8 T cell infiltration in spinal cords. We propose that GMME1 defines a new class of agents for the treatment of autoimmune ailments by selectively targeting lymphomyeloid cells expressing CCR2.

Differential effects of decoy chemokine (7ND) gene therapy on acute, biphasic and chronic autoimmune encephalomyelitis: implication for pathomechanisms of lesion formation.

Multiple sclerosis (MS) exhibits several clinical subtypes such as the relapsing-remitting (RR) and secondary progressive (SP) forms. In accordance with this, formation of demyelinating plaques in the central nervous system (CNS) occurs by different mechanisms. In the present study, we induced acute, biphasic and chronic (RR or SP) EAE in rats and examined the effects of decoy chemokine (7ND) gene therapy, which inhibits the migration of macrophages, to address the above issue. Interestingly, it was demonstrated that the clinical signs of acute EAE and the first attack of biphasic EAE were minimally affected, whereas chronic EAE and the relapse of biphasic EAE were completely suppressed with 7ND treatment. In the CNS, the number of infiltrating macrophages was reduced in all the stages of the three types of EAE. These findings suggest that in acute EAE and in the first attack of biphasic EAE, where anti-macrophage migration therapy was almost ineffective, pathogenic T cells are mainly involved in lesion formation. In contrast, the relapse of biphasic EAE and chronic EAE macrophages play a major role in the disease process. Thus, the mechanisms of lesion formation are not uniform and immunotherapy should be performed on the basis of information about the pathomechanisms of autoimmune diseases.

Effects of intratumoral injection of CCL2 on monocyte-endothelial cell interactions in mouse pancreatic cancer.

OBJECTIVE: Although monocyte infiltration is an important aspect of the host response to tumor growth, the mechanisms of recruitment and their impact on tumor growth are still unknown. The authors studied monocyte-endothelial interaction and the effect of chemokine CCL2 in orthotopic mouse pancreatic cancer. METHODS: The rolling and adhesion of labeled monocytes in peritumoral and intratumoral areas were assessed by using an intravital microscope. Further, the effects of intratumoral injection or superfusion of CCL2 on in situ recruitment of monocytes and other immune cells and adhesion molecules were investigated. RESULTS: More monocytes were recruited in the peritumoral area than in the intratumoral area with increased vascular interaction, and the effect was more apparent by intratumoral CCL2 injection than superfusion. In both CCL2-treated groups infiltration of CD11b(+), CD68(+), and CD4(+) cells were increased, but the magnitude of increase was larger in intratumoral injection. Quantitative RT-PCR for the tumor tissue revealed that ICAM-1 expression was increased by the injection of CCL2. CONCLUSION: These results show intratumoral injection of CCL2 induces effective interaction between monocytes and endothelial cells in the peritumoral area of pancreatic cancer accompanied by the upregulation of ICAM-1 and may possibly become a tool for immunotherapy by promoting the infiltration of immune cells in cancers.