Chemokines Macrophage Inflammatory Protein-1alpha Research Articles

The PPARα agonist fenofibrate reduces the cytokine imbalance in a maternal immune activation model of schizophrenia

Maternal infections during pregnancy may increase the risk of psychiatric disorders in offspring. We recently demonstrated that activation of peroxisome proliferator-activate receptor-α (PPARα), with the clinically available agonist fenofibrate (FEN), attenuates the neurodevelopmental disturbances induced by maternal immune activation (MIA) in rat offspring. We hypothesized that fenofibrate might reduce MIA-induced cytokine imbalance using a MIA model based on the viral mimetic polyriboinosinic-polyribocytidilic acid [poly (I:C)]. By using the Bio-Plex Multiplex-Immunoassay-System, we measured cytokine/chemokine/growth factor levels in maternal serum and in the fetal brain of rats treated with fenofibrate, at 6 and 24 h after poly (I:C). We found that MIA induced time-dependent changes in the levels of several cytokines/chemokines/colony-stimulating factors (CSFs). Specifically, the maternal serum of the poly (I:C)/control (CTRL) group showed increased levels of (i) proinflammatory chemokine macrophage inflammatory protein 1-alpha (MIP-1α), (ii) tumor necrosis factor-alpha (TNF-α), the monocyte chemoattractant protein-1 (MCP-1), the macrophage (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Conversely, in the fetal brain of the poly (I:C)/CTRL group, interleukin 12p70 and MIP-1α levels were lower than in vehicle (veh)/CTRL group. Notably, MIP-1α, TNF-α, keratinocyte derived chemokine (GRO/KC), GM-CSF, and M-CSF levels were lower in the poly (I:C)/FEN than in poly (I:C)/CTRL rats, suggesting the protective role of the PPARα agonist. PPARα might represent a therapeutic target to attenuate MIA-induced inflammation.

Ecklonia cava Extract Exerts Anti-Inflammatory Effect in Human Gingival Fibroblasts and Chronic Periodontitis Animal Model by Suppression of Pro-Inflammatory Cytokines and Chemokines.

Periodontitis is one of the most common chronic inflammatory diseases. The anti-inflammatory effect of the extract from brown algae Ecklonia cava was analyzed in lipopolysaccharide (LPS)-stimulated human gingival fibroblasts (HGF-1), the most abundant cells in gingival tissue. The gene expressions of cyclooxygenase-2 and interleukin-6 were decreased by 78 and 50%, respectively, at 100 μg/mL Ecklonia cava extract (ECE) treatment. The gene expressions of matrix metalloproteases (MMP-2 and MMP-8) and chemokines (macrophage inflammatory protein 1-alpha and stromal cell-derived factor 1) were also significantly down-regulated by ECE treatment (p < 0.05). The increased reactive oxygen species (ROS) production in HGF-1 cells by LPS stimulation was decreased by 30% at 100 μg/mL ECE treatment. The mitogen-activated protein kinase pathway and the nuclear factor-kappa B (NF-κB) signal activated by ROS were suppressed by ECE in a dose-dependent manner. ECE treatment (400 mg/kg, 8 weeks) significantly improved alveolar bone resorption in the ligature-induced chronic periodontitis rat model. ECE supplementation also lowered elevated mRNA expression of the receptor activator of nuclear factor-kappa B (RANKL)/osteoprotegerin (OPG) in the gingival tissue (p < 0.05). Therefore, ECE mitigated gingival tissue destruction and bone resorption associated with chronic periodontitis condition.

Abstract 316: Effect of schweinfurthins on malignant plasma cells

Abstract Multiple myeloma (MM), a bone marrow neoplasm is currently incurable and effective alternative therapies are needed. Schweinfurthin-3114 (S-3114) purified from a plant extract, inhibits growth of established MM cell lines. The mechanism(s) for this effect are not fully characterized. To advance understanding, MM cell lines RPMI-8226 and MM.1s were incubated with increasing concentrations of S-3114 for up to 48 hours. S-3114 decreased cell viability (trypan blue assay) and mitochondrial activity (MTT assay) in a concentration and time-dependent manner (p≤0.05). RPMI-8226 cells were more sensitive (EC50 2.36 nM) than MM.1s (EC50 11.1 nM) at 48 hours. Flow cytometry analyses revealed increased apoptosis in a concentration and time-dependent manner in both cell lines (64.0% in RPMI-8226 with 20 nM S-3114; 86.3% in MM.1s with 270 nM S-3114, both at 48 hours). S-3114 also induced cell cycle arrest at G2/M. In RPMI-8226, the percent of cells arrested in G2/M increased from 45.9 to 66.1 with 3.5 nM S-3114 at 24 and 48 hours, respectively. In RPMI-8226, 20 nM S-3114 for 24 and 48 hours caused a 100% G2/M arrest. In both cell lines, S-3114 reduced protein kinase B (Akt) activation (Western blot) in a concentration and time-dependent manner. In MM.1s the apoptotic extrinsic pathway (caspase 8), and not the intrinsic pathway (caspase 9), is highly involved. In fact, S-3114 (20 nM for 48 hours, and 270 nM for 24 and 48 hours) caused a major cleavage for both 43 and 18 KDa caspase 8 fragments. Also cleaved Poly ADP-Ribose Polymerase 1 (PARP-1) increased at S-3114 concentrations of 20 and 270 nM at 24 and 48 hours. S-3114 influence on Golgi-mediated secretion was evaluated by measuring the soluble chemokine Macrophage Inflammatory Protein-1 alpha (MIP-1α), which is secreted by the Golgi and it is involved in the progression of MM disease. S-3114 treatment for 48 hours stimulated the MIP-1α negative cell line (RPMI-8226) to significantly increase MIP-1α RNA levels and to secrete MIP-1α at low concentrations. On the contrary, despite that prolonged S-3114 treatment significantly increased MIP-1α RNA levels, its secretion is decreased in the MIP-1α positive cell line (MM.1s). These in vitro studies demonstrate that S-3114 impairs the Phosphoinositide 3-Kinases (PI3K)/Akt signaling pathway resulting in cell death and/or cell cycle arrest. Data also shows an increased gene expression of the pro-survival MIP-1α by S-3114. Our ongoing studies are focused on schweinfurthin effects on MIP-1 α and Golgi secretion. We postulate that the cytotoxic impact of S-3114 requires disruption of MIP-1α signaling to regulate cell cycle, survival, and impairment of Golgi architecture and secretion in malignant plasma cells. In aggregate, these results reveal mechanism(s) for S-3114 effects and support further work advancing S-3114 as a novel therapy for MM. Citation Format: Barbara Manfredi, Raymond J. Hohl. Effect of schweinfurthins on malignant plasma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 316.

881TiP A multi-centre, open-label phase II trial of the combination of VB10.16 and atezolizumab in patients with advanced or recurrent, non-resectable HPV16 positive cervical cancer

HPV16 accounts for almost 60% of the cervical cancer cases. There is an urgent need to develop therapeutic HPV vaccines for control of chronic HPV infection and associated malignancies. HPV16 oncogenes E6/E7 are truly cancer-specific viral antigens and represent ideal targets for a therapeutic HPV16 vaccine. VB10.16 is a targeted DNA-based immunotherapy designed to treat HPV16-associated pre- and malignant lesions. The DNA vaccine encodes a recombinant protein consisting of mutation-inactivated E6 and E7 proteins linked to the natural human chemokine macrophage inflammatory protein-1 alpha (MIP-1α).

Identifying a Novel Role for Fractalkine (CX3CL1) in Memory CD8+ T Cell Accumulation in the Omentum of Obesity-Associated Cancer Patients.

The omentum is enriched with pro-inflammatory effector memory CD8+ T cells in patients with the obesity-associated malignancy, esophagogastric adenocarcinoma (EAC) and we have identified the chemokine macrophage inflammatory protein-1alpha as a key player in their active migration to this inflamed tissue. More recently, others have established that subsets of memory CD8+ T cells can be classified based on their surface expression of CX3CR1; the specific receptor for the inflammatory chemokine fractalkine. CD8+ T cells expressing intermediate levels (CX3CR1INT) are defined as peripheral memory, those expressing the highest levels (CX3CR1HI) are effector memory/terminally differentiated and those lacking CX3CR1 (CX3CR1NEG) are classified as central memory. To date, the fractalkine:CX3CR1 axis has not been examined in the context of CD8+ T cell enrichment in the omentum and here we examine this chemokines involvement in the accumulation of memory CD8+ T cells in the omentum of EAC patients. Our data show that fractalkine is significantly enriched in the omentum of EAC patients and drives migration of T cells derived from EAC patient blood. Furthermore, CX3CR1 is endocytosed specifically by CD8+ T cells upon encountering fractalkine, which is consistent with the significantly diminished frequencies of CX3CR1INT and CX3CR1HI CD8+ T cells in the fractalkine-rich environment of omentum in EAC, relative to matched blood. Fractalkine-mediated endocytosis of CX3CR1 by CD8+ T cells is sustained and is followed by enhanced surface expression of L-selectin (CD62L). These novel data align with our findings that circulating CX3CR1NEG CD8+ T cells express higher levels of L-selectin than CX3CR1INT CD8+ T cells. This is consistent with previous reports and implicates fractalkine in the conversion of CX3CR1INT CD8+ T cells to a CX3CR1NEG phenotype characterized by alterations in the migratory capacity of these T cells. For the first time, these findings identify fractalkine as a driver of T cell migration to the omentum in EAC and indicate that CD8+ T cells undergo sequenced fractalkine-mediated alterations in CX3CR1 and L-selectin expression. These data implicate fractalkine as more than a chemotactic cytokine in obesity-associated meta-inflammation and reveal a role for this chemokine in the maintenance of the CX3CR1NEG CD8+ T cell populations.

AAV-mediated gene delivery attenuates neuroinflammation in feline Sandhoff disease

Sandhoff disease (SD) is a lysosomal storage disorder characterized by the absence of hydrolytic enzyme β-N-acetylhexosaminidase (Hex), which results in storage of GM2 ganglioside in neurons and unremitting neurodegeneration. Neuron loss initially affects fine motor skills, but rapidly progresses to loss of all body faculties, a vegetative state, and death by five years of age in humans. A well-established feline model of SD allows characterization of the disease in a large animal model and provides a means to test the safety and efficacy of therapeutic interventions before initiating clinical trials. In this study, we demonstrate a robust central nervous system (CNS) inflammatory response in feline SD, primarily marked by expansion and activation of the microglial cell population. Quantification of major histocompatibility complex II (MHC-II) labeling revealed significant up-regulation throughout the CNS with areas rich in white matter most severely affected. Expression of the leukocyte chemokine macrophage inflammatory protein-1 alpha (MIP-1α) was also up-regulated in the brain. SD cats were treated with intracranial delivery of adeno-associated viral (AAV) vectors expressing feline Hex, with a study endpoint 16weeks post treatment. AAV-mediated gene delivery repressed the expansion and activation of microglia and normalized MHC-II and MIP-1α levels. These data reiterate the profound inflammatory response in SD and show that neuroinflammation is abrogated after AAV-mediated restoration of enzymatic activity.

RANDOMIZATION OF PATIENTS WITH GLIOBLASTOMA TO VACCINE SITE PRE-CONDITIONING WITH TETANUS-DIPHTHERIA TOXOID SYSTEMICALLY ENHANCES MIGRATION AND THERAPEUTIC EFFECT OF CYTOMEGALOVIRUS PP65-PULSED DENDRITIC CELL VACCINE IN A MIP-1 -DEPENDENT FASHION

BACKGROUND: Dendritic cell (DC) vaccine efficacy is limited by suboptimal migration to vaccine site-draining lymph nodes (VDLNs). In mice, vaccine site conditioning with inflammatory cytokines or mature DCs increases DC trafficking and the induction of antigen-specific T cells. We assessed the impact of DC migration to VDLNs on clinical outcomes in patients with newly-diagnosed glioblastoma (GBM) by randomizing patients to one of two vaccine site conditioning strategies. METHODS: We compared effects of vaccine site conditioning with either unpulsed DCs or tetanus-diphtheria (Td) toxoid prior to a vaccine of 111Indium-labeled DCs and monitored subsequent migration to VDLNs using SPECT/CT imaging. Clinical responses following vaccine site conditioning before DC vaccination were evaluated with progression-free survival (PFS) and overall survival (OS). A preclinical model using DCs derived from fluorescent transgenic mice was employed to explore DC migration to VDLNs in the context of vaccine site conditioning. The capacity for increased DC migration to induce tumor-specific immune responses in mice was evaluated. RESULTS: In patients with GBM, migration of injected DCs to VDLNs via Td conditioning was significantly increased compared to controls (P = 0.04), and the extent of migration was strongly associated with clinical outcomes (HR = 0.845 P = 0.027 for PFS; HR = 0.820 P = 0.023 for OS; Cox proportional hazards model). Furthermore, patients randomized to Td demonstrated improved OS compared to controls (median OS 18.5 months vs >32.5 months, P = 0.013). In accordance with our clinical observations, our preclinical model showed that Td conditioning significantly increased DC migration systemically to VDLNs (P = 0.0001). Td conditioning resulted in a significant elevation in serum levels of the chemokine macrophage inflammatory protein-1 alpha (MIP-1α) (P = 0.028). In vitro chemotaxis assays revealed a selective increase in human and murine DCs migrating towards serum from Td-conditioned patients and mice compared to controls. Furthermore, this preferential migration was severely abrogated with MIP-1α blockade (P = 0.02). In mice, Td conditioning in MIP-1α knockout mice only induced baseline migration efficiency compared to controls (P = 0.003). Td conditioning in mice dramatically suppressed tumor growth and improved DC vaccines in a tumor antigen-specific manner (P = 0.0001). Moreover, Td-conditioned MIP-1α knockout mice demonstrated significantly increased tumor burden compared to that of normal mice receiving Td prior to DC vaccination (P < 0.05). CONCLUSIONS: Td conditioning increased DC migration and tumor responses in a preclinical murine model corroborating observations from our phase I/II clinical trial. Our studies demonstrate a relationship between dendritic cell trafficking and antitumor efficacy and highlight MIP-1α as an important chemokine for improving dendritic cell immunotherapy. SECONDARY CATEGORY: Clinical Neuro-Oncology.

Neonatal cytokines and chemokines and risk of Autism Spectrum Disorder: the Early Markers for Autism (EMA) study: a case-control study

BackgroundBiologic markers of infection and inflammation have been associated with Autism Spectrum Disorders (ASD) but prior studies have largely relied on specimens taken after clinical diagnosis. Research on potential biologic markers early in neurodevelopment is required to evaluate possible causal pathways and screening profiles.ObjectiveTo investigate levels of cytokines and chemokines in newborn blood specimens as possible early biologic markers for autism.MethodsWe conducted a population-based case-control study nested within the cohort of infants born from July 2000 to September 2001 to women who participated in the prenatal screening program in Orange County, California, USA. The study population included children ascertained from the California Department of Developmental Services with Autism Spectrum Disorder (ASD, n = 84), or developmental delay but not ASD (DD, n = 49), and general population controls randomly sampled from the birth certificate files and frequency matched to ASD cases on sex, birth month and birth year (GP, n = 159). Cytokine and chemokine concentrations were measured in archived neonatal blood specimens collected for routine newborn screening.ResultsCytokines were not detected in the vast majority of newborn samples regardless of case or control status. However, the chemokine monocyte chemotactic protein-1 (MCP-1) was elevated and the chemokine Regulated upon Activation Normal T-Cell Expressed and Secreted (RANTES) was decreased in ASD cases compared to GP controls. The chemokines macrophage inflammatory protein-1alpha (MIP-1α) and RANTES were decreased in children with DD compared to GP controls.ConclusionMeasurement of immune system function in the first few days of life may aid in the early identification of abnormal neurodevelopment and shed light on the biologic mechanisms underlying normal neurodevelopment.

Hepatic macrophage migration and differentiation critical for liver fibrosis is mediated by the chemokine receptor C-C motif chemokine receptor 8 in mice

Chemokines critically control the infiltration of immune cells upon liver injury, thereby promoting hepatic inflammation and fibrosis. The chemokine receptor CCR8 can affect trafficking of monocytes/macrophages, monocyte-derived dendritic cells (DCs) and T-helper cell (Th) subsets, but its role in liver diseases is currently unknown. To investigate the functional role of CCR8 in liver diseases, ccr8(-/-) and wild-type (WT) mice were subjected to chronic experimental injury models of carbon tetrachloride (CCl(4) ) administration and surgical bile duct ligation (BDL). CCR8 was strongly up-regulated in the injured liver. Ccr8(-/-) mice displayed attenuated liver damage (e.g., ALT, histology, and TUNEL) compared to WT mice and were also protected from liver fibrosis in two independent injury models. Flow cytometry revealed reduced infiltrates of liver macrophages, neutrophils and natural killer cells, whereas hepatic CD4(+) T cells increased. The main CCR8-expressing cells in the liver were hepatic macrophages, and CCR8 was functionally necessary for CCL1-directed migration of inflammatory but not for nonclassical monocytes into the liver. Moreover, the phenotype of liver macrophages from injured ccr8(-/-) animals was altered with increased expression of DC markers and enhanced expression of T-cell-attracting chemokine macrophage inflammatory protein 1-alpha (MIP-1α/CCL3). Correspondingly, hepatic CD4(+) T cells showed increased Th1 polarization and reduced Th2 cells in CCR8-deficient animals. Liver fibrosis progression, but also subsequent T-cell alterations, could be restored by adoptively transferring CCR8-expressing monocytes/macrophages into ccr8(-/-) mice during experimental injury. CCR8 critically mediates hepatic macrophage recruitment upon injury, which subsequently shapes the inflammatory response in the injured liver, affecting macrophage/DC and Th differentiation. CCR8 deficiency protects the liver against injury, ameliorating initial inflammatory responses and hepatic fibrogenesis. Inhibition of CCR8 or its ligand, CCL1, might represent a successful therapeutic target to limit liver inflammation and fibrosis progression.

Type 1 T-cell responses in chlamydial lung infections are associated with local MIP-1α response

Chemokines and their receptors are important mediators of leukocyte trafficking and recruitment and sometimes work as modulators of T-cell responses during infections and inflammation. Modulating the biological activity of chemokines has been found to influence the course of diseases. However, little is known about the role of chemokine responses during chlamydial lung infections. We therefore analyzed the dynamics of multiple chemokines, which are frequently associated with type 1 (Th1) T cell immune responses, and their receptors for their expression in the lungs during Chlamydia muridarum (Cm) infections. We also examined the relationship between chemokine responses and the development of Th1 responses as well as the clearance of infection. Our results showed that in parallel with the high levels of gamma interferon (IFN-gamma) and IL-12 production in the lungs and draining lymph nodes, and the expansion of IFN-gamma-producing CD4 and CD8+ T cells, the production of the cell-related chemokines RANTES, IFN-gamma-inducible protein-10 (IP-10) and macrophage inflammatory protein-1alpha (MIP-1alpha) and their receptor CCR1 was elevated in the lung tissues after infection. Interestingly, in a later phase of infection, the expression of RANTES and IP-10 remained elevated but the expression of MIP-1alpha and CCR1 decreased to a low level, which suggests a closer association with the pattern of Th1 cytokine responses in the process of infection. These results suggest a close association between the MIP-1alpha response and the Th1-type T-cell responses in chlamydial lung infections.

The roles of chemokines in rabies virus infection: overexpression may not always be beneficial.

It was found previously that induction of innate immunity, particularly chemokines, is an important mechanism of rabies virus (RABV) attenuation. To evaluate the effect of overexpression of chemokines on RABV infection, chemokines macrophage inflammatory protein 1alpha (MIP-1alpha), RANTES, and IP-10 were individually cloned into the genome of attenuated RABV strain HEP-Flury. These recombinant RABVs were characterized in vitro for growth properties and expression of chemokines. It was found that all the recombinant viruses grew as well as the parent virus, and each of the viruses expressed the intended chemokine in a dose-dependent manner. When these viruses were evaluated for pathogenicity in the mouse model, it was found that overexpression of MIP-1alpha further decreased RABV pathogenicity by inducing a transient innate immune response. In contrast, overexpression of RANTES or IP-10 increased RABV pathogenicity by causing neurological diseases, which is due to persistent and high-level expression of chemokines, excessive infiltration and accumulation of inflammatory cells in the central nervous system, and severe enhancement of blood-brain barrier permeability. These studies indicate that overexpression of chemokines, although important in controlling virus infection, may not always be beneficial to the host.

Expression of cytokines and chemokines and microvasculature alterations of the tongue from patients with chronic Chagas’ disease

Chagas' disease is caused by the protozoan Trypanosoma cruzi and continues to be a significant public health problem, since 10 million people are still infected in Latin America. The purpose of this study was to analyze the microvasculature alterations as well the expression of cytokines and chemokines in the tongues from patients with chronic Chagas' disease (CC; n = 18), comparatively with a non-chagasic group (NC; n = 22). We observed several vascular alterations in the tongue of CC such as a greater vascular diameter, increased vascular wall area, high density of the blood vessels, and increased thickening of the capillary basement membrane. The expression of cytokines interferon gamma and tumor necrosis factor alpha and chemokine macrophage inflammatory protein 1alpha were significantly down-regulated in the tongue of CC group. These results demonstrated that, in the tongue of chagasic patients, a microvascular abnormality and immunological impairment occurs, probably due to chronic inflammation evoked by T. cruzi antigens.

GABAergic activities enhance macrophage inflammatory protein‐1α release from microglia (brain macrophages) in postnatal mouse brain

Microglial cells (brain macrophages) invade the brain during embryonic and early postnatal development, migrate preferentially along fibre tracts to their final position and transform from an amoeboid to a ramified morphology. Signals by which the invading microglia communicate with other brain cells are largely unknown. Here, we studied amoeboid microglia in postnatal corpus callosum obtained from 6- to 8-day-old mice. These cells accumulated on the surface of acute brain slices. Whole-cell patch-clamp recordings revealed that the specific GABA(A) receptor agonist muscimol triggered a transient increase in conductance typical for inward rectifying potassium channels in microglia. This current increase was not mediated by microglial GABA(A) receptors since microglial cells removed from the slice surface no longer reacted and cultured microglia only responded when a brain slice was placed in their close vicinity. Muscimol triggered a transient increase in extracellular potassium concentration ([K(+)](o)) in brain slices and an experimental elevation of [K(+)](o) mimicked the muscimol response in microglial cells. Moreover, in adult brain slices, muscimol led only to a minute increase in [K(+)](o) and microglial cells failed to respond to muscimol. In turn, an increase in [K(+)](o) stimulated the release of chemokine macrophage inflammatory protein-1alpha (MIP1-alpha) from brain slices and from cultures of microglia but not astrocytes. Our observations indicate that invading microglia in early postnatal development sense GABAergic activities indirectly via sensing changes in [K(+)](o) which results in an increase in MIP1-alpha release.

Mycobacterium abscessus and M. avium trigger Toll-like receptor 2 and distinct cytokine response in human cells.

Mycobacterium avium (MAV) and M. abscessus (MAB) are ubiquitous environmental organisms increasingly recognized to cause chronic lung disease in patients with apparently normal immune function. Little is yet known about their human pathophysiology. Our objective was to examine cytokine and chemokine responses (protein and gene expression) and signaling pathways triggered by reference and clinical isolates of MAB and MAV in human peripheral blood mononuclear cells, monocytes, and murine bone marrow-derived macrophages in vitro. MAB-induced TNF-alpha production was higher than that induced by MAV. IFN-gamma, IL-1beta, and the chemokines macrophage inflammatory protein-1alpha and regulated on activation, normal T cell expressed and secreted were equally up-regulated. Differences between MAB and MAV do not require replication and are heat stable. We found no differential effect due to rough or smooth colonies within the same species. Similar to MAV, MAB triggered mitogen-activated protein kinase (MAPK) signaling and nuclear factor-kappaB translocation. Induction of TNF-alpha was dependent on MAPK pathways, since pre-incubation of cells with signaling inhibitors led to more than 85% reduction in cytokine secretion. MAB also triggered a Toll-like receptor 2 (TLR2)-mediated response that led to TNF-alpha production by human monocytes. Accordingly, stimulation of murine TLR2- or myeloid differentiation factor 88-deficient bone marrow-derived macrophages did not elicit TNF-alpha, reinforcing a critical role for TLR2 in MAB-induced cell activation. We concluded that MAB signals human cells through MAPK and TLR2 pathways and triggers more pronounced pro-inflammatory cytokines and chemokines than MAV.

Priming of CD4+ T cells with porin of Shigella dysenteriae activates the cells toward type 1 polarization

Macrophages treated with porin of Shigella dysenteriae were potent stimulators of naive T(h) cells since the porin-pulsed macrophages strongly proliferated the T cells and up-regulated the activation molecules CD69 and CD25 on CD4(+) T cells in allogeneic mixed leukocyte reaction. Immunization of C57BL/6 mice with porin selectively induced the intracellular expression and release of IFN-gamma and had no effect on IL-4 expression. In parallel to the predominant release of the T(h)1 cytokine IFN-gamma, up-regulation of CCR5 on the immune CD4(+) T cells and messenger ribonucleic acid (mRNA) expression of the T cell chemokines macrophage inflammatory protein-1 alpha and regulated on activation normal T cell expressed and secreted showed T(h)1 bias. The porin-primed CD4(+) T cells expressed the mRNA for T-box expressed in T cells, a T(h)1-specific transcription factor confirming the adjuvant-induced transition of T cells to polarized effector T(h)1 cells. The immune CD4(+) T cells proliferated and released IL-2 and IFN-gamma profoundly in response to re-challenge with porin-pulsed macrophages but not to BSA-pulsed macrophages in vitro, which demonstrated the presence of porin-specific CD4(+) T cells of T(h)1 phenotype. The study highlights that porin has the capacity of an adjuvant to unfold and maintain the T cell function and thereby to activate adaptive immunity.

Influence of Coenzyme Q10 on release of pro‐inflammatory chemokines in the human monocytic cell line THP‐1

Coenzyme Q_{10} (CoQ_{10}) is an obligatory element in the mitochondrial electron transport system and functions as a potent antioxidant of lipid membranes. In-vivo and in-vitro studies indicate an involvement of CoQ_{10} in inflammatory pathways. Here we studied in the human monocytic cell-line THP-1 the influence of CoQ_{10} on LPS-induced secretion of the pro-inflammatory chemokines Macrophage inflammatory protein-1 alpha (MIP-1alpha), Regulated upon activation, normal T cell expressed and secreted (RANTES) and Monocyte chemoattractant protein-1 (MCP-1). In comparison to unstimulated cells, LPS leads to 22-, 3- and 4.5-fold higher levels of MIP-1alpha, RANTES and MCP-1 in the cell culture medium, respectively. Pre-incubation of cells with 10 microM CoQ_{10} resulted in a significant decrease of LPS-induced MIP-1alpha and RANTES secretion to 55.04% (p = 0.02) and 76.84% (p = 0.04), respectively. In conclusion, CoQ_{10} reduces the LPS-induced secretion levels of the pro-inflammatory chemokines MIP-1alpha and RANTES in the human monocytic cell line THP-1. These data suggest that CoQ_{10} possesses anti-inflammatory properties.

STATINS REDUCE MACROPHAGE INFLAMMATORY PROTEIN‐1α EXPRESSION IN HUMAN ACTIVATED MONOCYTES

1. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) exhibit a wide variety of anti-atherogenic effects that may be independent of their property to lower plasma cholesterol. 2. In order to systematically investigate these effects at a cellular level, we investigated gene expression in phorbol myristate acetate (PMA)-activated and non-activated human THP-1 monocytes in response to statins using cDNA arrays. 3. Of 588 genes tested, 26 were differentially expressed in the presence of statins. A marked reduction was found for the chemokine macrophage inflammatory protein-1alpha (MIP-1alpha). The decrease in MIP-1alpha mRNA expression after incubation with statins was confirmed by quantitative reverse transcription-polymerase chain reaction in THP-1 monocytes and human freshly isolated monocytes. Macrophage inflammatory protein-1alpha protein in THP-1 monocytes was reduced from 377 to 299 and 305 pg/mL by 0.1 micro mol/L simvastatin and 0.01 micro mol/L cerivastatin, respectively. The reduction in MIP-1alpha expression by statins was due, at least in part, to transcriptional inhibition of MIP-1alpha promoter activity. 4. The CC receptor ligand MIP-1alpha is a chemokine that has been implicated in atherosclerotic lesion formation. The present findings suggest that statin-mediated immunomodulation by inhibiting MIP-1alpha could contribute to the beneficial effects of statin therapy independent of lowering plasma cholesterol.

Erratum for vol. 89, p. 3582

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and Steel factor (SLF) synergistically stimulate Raf-1 kinase activity, protein synthesis, and proliferation in hematopoietic MO7e cells; synergistic action of these factors is blocked by the suppressive chemokines macrophage inflammatory protein-1alpha (MIP-1alpha) and interferon-inducible protein 10 (IP-10; Aronica et al, J Biol Chem 270:21998, 1995). We assessed the potential for both stimulatory and inhibitory factors to act through the MAP kinase signaling pathway by studying the effects of growth factors and chemokines on MAP kinase activation. Also, because activation of kinase signaling pathways and stimulation of protein synthesis by peptide growth factors are associated with increased phosphorylation of eukaryotic initiation factor 4E (elF-4E) and the translational repressor 4E-binding protein 1 (4E-BP1) in some target cells, we investigated whether growth factor treatment could alter eIF-4E or 4E-BP1 phosphorylation state in MO7e cells. We report that treatment of MO7e cells with GM-CSF and SLF stimulated significant, greater-than-additive increases in MAP kinase activity and the phosphorylation of both eIF-4E and 4E-BP1. Increased 4E-BP1 phosphorylation correlated with a decrease in the association of 4E-BP1 with eIF-4E. Growth factor-induced phosphorylation of 4E-BP1 and dissociation of 4E-BP1 from eIF-4E was blocked in cells treated with rapamycin, wortmannin, or PD098059. Treatment of cells with IP-10 or MIP-1alpha blocked the stimulatory effects of GM-CSF and SLF, resulting in suppression of MAP kinase activity, eIF-4E and 4E-BP1 phosphorylation, and eIF-4E/4E-BP1 dissociation. Our results suggest that GM-CSF and SLF exert part of their combined growth-promoting effects on MO7e cells through activation of MAP kinase and enhancement of eIF-4E and 4E-BP1 phosphorylation and dissociation and that suppression of growth factor-induced protein synthesis by MIP-1alpha and IP-10 involves translational repression at the level of eIF-4E.

Transcription factor T-bet regulates inflammatory arthritis through its function in dendritic cells

The transcription factor T-bet (Tbx21) plays a major role in adaptive immunity and is required for optimal IFN-gamma production by DCs. Here we demonstrate an essential function for T-bet in DCs in controlling inflammatory arthritis. We show that collagen antibody-induced arthritis (CAIA), a model of human RA, is a bipartite disease characterized by an early innate immune system component intact in RAG2 mice and a later adaptive immune system phase. Mice lacking T-bet had markedly reduced joint inflammation at both early and late time points and RAG2T-bet double-deficient mice were essentially resistant to disease. Remarkably, adoptive transfer of T-bet-expressing DCs reconstituted inflammation in a T-bet deficient and T-bet/RAG2-deficient milieu. T-bet regulates the production of proinflammatory cytokine IL-1alpha and chemokines macrophage inflammatory protein-1alpha (MIP-1alpha) and thymus- and activation-related chemokine (TARC) by DCs. Further, T-bet expression in DCs is required for T helper cell activation. We conclude that T-bet plays a vital function in DCs that links innate and adaptive immunity to regulate inflammatory responses. T-bet provides an attractive new target for the development of novel therapeutics for inflammatory arthritis.

Amelioration of rat adjuvant‐induced arthritis by Met‐RANTES

CC chemokines and their receptors play a fundamental role in trafficking and activation of leukocytes at sites of inflammation, contributing to joint damage in rheumatoid arthritis. Met-RANTES, an amino-terminal-modified methionylated form of RANTES (CCL5), antagonizes the binding of the chemokines RANTES and macrophage inflammatory protein 1alpha (MIP-1alpha; CCL3) to their receptors CCR1 and CCR5, respectively. The aim of this study was to investigate whether Met-RANTES could ameliorate adjuvant-induced arthritis (AIA) in the rat. Using immunohistochemistry, enzyme-linked immunosorbent assay, real-time reverse transcription-polymerase chain reaction, Western blot analysis, adoptive transfer, and chemotaxis, we defined joint inflammation, bony destruction, neutrophil and macrophage migration, Met-RANTES binding affinity to rat receptors, proinflammatory cytokine and bone marker levels, CCR1 and CCR5 expression and activation, and macrophage homing into joints with AIA. Administration of Met-RANTES as a preventative reduced the severity of joint inflammation. Administration of Met-RANTES to ankles with AIA showed decreases in inflammation, radiographic soft tissue swelling, and bone erosion. Met-RANTES significantly reduced the number of neutrophils and macrophages at the peak of arthritis compared with saline-injected controls. Competitive chemotaxis in peripheral blood mononuclear cells demonstrated that Met-RANTES inhibited MIP-1alpha and MIP-1beta at 50% inhibition concentrations of 5 nM and 2 nM, respectively. Furthermore, levels of tumor necrosis factor alpha, interleukin-1beta, macrophage colony-stimulating factor, and RANKL were decreased in joints with AIA in the Met-RANTES group compared with the control group. Interestingly, the expression and activation of CCR1 and CCR5 in the joint were down-regulated in the Met-RANTES group compared with the control group. Functionally, Met-RANTES administration decreased adoptively transferred peritoneal macrophage homing into the joint. The data suggest that the targeting of Th1-associated chemokine receptors reduce joint inflammation, bone destruction, and cell recruitment into joints with AIA.