CCL3, IL-7, IL-13 and IFNγ transcripts are increased in skin's biopsy of systemic sclerosis.

Although several cytokines and chemokines have been investigated as possible mediators of fibrosis in systemic sclerosis (SSc), specific correlation between cytokines and organ involvement have not been found yet, and a cytokine profile characteristic of SSc is far to be identified. We studied the profile of antifibrotic and profibrotic transcripts involved in skin of SSc patients. The mRNA expression was detected by fluorescence-based quantitative real-time PCR (qPCR) in skin's biopsies from 14 patients with SSc and 5 healthy controls. PDGF-A, CTGF, CCL3, IL-6, IL-13, IL-7, IFNγ, IL-17, IL-22 and RORc were analysed in these samples. CCL3, IL-7, IL-13 and IFN-γ were more expressed in skin's biopsy of patients with SSc (P = 0.0002, P = 0.0082, P = 0.0243, P = 0.0335, respectively) when compared with healthy controls. We also found a positive correlation between CCL3 and IL-7 transcripts (P = 0.0050 r = 0.7187). Furthermore, we observed that patients with lung involvement had lower expression of PDGF-A (P = 0.0385). We found an increase in IL-7, IFN-γ, CCL3 and IL-13 relative mRNA expressions on the skin's biopsy of patients with SSc, and a positive correlation between IL-7 and CCL3. These molecules are involved in the pathogenesis of SSc, and how their interactions occur should be the subject of further studies.

MIP-1α/CCL3-expressing basophil-lineage cells drive the leukemic hematopoiesis of chronic myeloid leukemia in mice.

Basophilia is a frequently observed hematological abnormality in chronic myeloid leukemia (CML), but its pathophysiological roles are undefined. We previously demonstrated that an inflammatory chemokine, CCL3, preferentially acts on normal hematopoietic stem/progenitor cells and crucially contributes to the maintenance of leukemia initiating cells (LICs) in bone marrow (BM) during the initiation process of CML. However, the major cellular source of CCL3 in BM and the precise mechanism of CCL3-mediated maintenance of LICs remain to be investigated. To delineate the cellular process facilitating this CCL3-mediated crosstalk between normal and leukemic hematopoiesis, we precisely examined CCL3-expressing cells and their functions in both normal hematopoiesis and CML leukemogenesis. Herein, we demonstrate that basophils can constitutively express CCL3 to negatively regulate the normal hematopoietic process, especially hematopoietic reconstitution after BM transplantation. Moreover, CCL3-expressing basophil-like leukemia cells were found to accumulate in CML BM and supported the predominant expansion of LICs therein. These observations suggest that intra-BM basophil expansion can favor leukemia-tropic hematopoiesis in CML by providing CCL3, a potent inhibitor of normal hematopoiesis and that basophil-derived CCL3 may be a novel target molecule for the treatment of CML.

Mast cell-mediated antigen presentation regulates CD8+ T cell effector functions.

The characteristics, importance, and molecular requirements for interactions between mast cells (MCs) and CD8(+) T cells have not been elucidated. Here, we demonstrated that MCs induced antigen-specific CD8(+) T cell activation and proliferation. This process required direct cell contact and MHC class I-dependent antigen cross-presentation by MCs and induced the secretion of interleukin-2, interferon-gamma, and macrophage inflammatory protein-1alpha by CD8(+) T cells. MCs regulated antigen-specific CD8(+) T cell cytotoxicity by increasing granzyme B expression and by promoting CD8(+) T cell degranulation. Because MCs also upregulated their expression of costimulatory molecules (4-1BB) and released osteopontin upon direct T cell contact, MC-T cell interactions probably are bidirectional. In vivo, adoptive transfer of antigen-pulsed MCs induced MHC class I-dependent, antigen-specific CD8(+) T cell proliferation, and MCs regulated CD8(+) T cell-specific priming in experimental autoimmune encephalomyelitis. Thus, MCs are important players in antigen-specific regulation of CD8(+) T cells.

P2Y12 receptors in primary microglia activate nuclear factor of activated T-cell signaling to induce C-C chemokine 3 expression.

Microglia are widely accepted as surveillants in the central nervous system that are continually searching the local environment for signs of injury. Following an inflammatory situation, microglia alter their morphology, extend ramified processes, and undergo cell body hypertrophy. Extracellular nucleotides are recognized as a danger signal by microglia. ADP acting on P2Y12 receptors induce process extension of microglia thereby attracting microglia to the site of adenosine tri-phosphate/ADP leaking or release. However, the question whether ADP/P2Y12 receptor signaling directly stimulates the production or release of inducible factors such as cytokines remains unclear. In this study, we found that CC chemokine ligand 3 (CCL3) is induced by ADP-treated primary microglia. Pharmacological characterization using pertussis toxin, a P2Y12 receptor inhibitor, and a calcium chelator revealed that CCL3 induction was caused by P2Y12 receptor-mediated intracellular calcium elevation. Next, nuclear factor of activated T-cell dephosphorylation and nuclear translocalization were observed. Calcineurin, an inhibitor for nuclear factor of activated T cell, suppressed CCL3 induction. These data indicate that microglial P2Y12 receptors are utilized to trigger an acute inflammatory response in microglia via rapid CCL3 induction after ADP stimulation.

Disseminated Cryptococcosis Due to Anti-Granulocyte-Macrophage Colony-Stimulating Factor Autoantibodies in the Absence of Pulmonary Alveolar Proteinosis.

INTRODUCTION: Autoantibodies to granulocyte-macrophage colony-stimulating factor (GM-CSF) can cause acquired pulmonary alveolar proteinosis (PAP). Cases of acquired PAP susceptible to typical respiratory pathogens and opportunistic infections have been reported. Anti-GM-CSF autoantibodies have been reported in a few patients with cryptococcal meningitis. This study evaluated the presence of neutralizing anti-GM-CSF autoantibodies in patients without known congenital or acquired immunodeficiency with severe pulmonary or extrapulmonary cryptococcal infection but without PAP. METHODS: We took a clinical history and performed an immunologic evaluation and screening of anti-cytokine autoantibodies in patients with cryptococcal meningitis. The impact of autoantibodies to GM-CSF on immune function was assessed by intracellular staining of GM-CSF-induced STAT5 phosphorylation and MIP-1α production in normal peripheral blood mononuclear cells incubated with plasma from patients or normal control subjects. RESULTS: Neutralizing anti-GM-CSF autoantibodies were identified in four patients with disseminated cryptococcosis, none of whom exhibited PAP. Plasma from patients blocked GM-CSF signaling and inhibited STAT5 phosphorylation and production of MIP-1α. One patient died of disseminated cryptococcosis involving the central nervous system, which was associated with defective GM-CSF activity. CONCLUSIONS: Anti-GM-CSF autoantibodies increase susceptibility to cryptococcal infection in adults without PAP. Cryptococcal central nervous system infection associated with anti-GM-CSF autoantibodies could result in neurological sequelae or be life-threatening. Therefore, timely detection of neutralizing anti-GM-CSF autoantibodies and development of an effective therapy are necessary to prevent deterioration of cryptococcal infection in these patients.

Didox (3,4-dihydroxybenzohydroxamic acid) suppresses IgE-mediated mast cell activation through attenuation of NFκB and AP-1 transcription.

Mast cell activation via the high-affinity IgE receptor (FcεRI) elicits production of inflammatory mediators central to allergic disease. As a synthetic antioxidant and a potent ribonucleotide reductase (RNR) inhibitor, Didox (3,4-dihyroxybenzohydroxamic acid) has been tested in clinical trials for cancer and is an attractive therapeutic for inflammatory disease. We found that Didox treatment of mouse bone marrow-derived mast cells (BMMC) reduced IgE-stimulated degranulation and cytokine production, including IL-6, IL-13, TNF and MIP-1a (CCL3). These effects were consistent using BMMC of different genetic backgrounds and peritoneal mast cells. While the RNR inhibitor hydroxyurea had little or no effect on IgE-mediated function, high concentrations of the antioxidant N-acetylcysteine mimicked Didox-mediated suppression. Furthermore, Didox increased expression of the antioxidant genes superoxide dismutase and catalase, and suppressed DCFH-DA fluorescence, indicating reduced reactive oxygen species production. Didox effects were not due to changes in FcεRI expression or cell viability, suggesting it inhibits signaling required for inflammatory cytokine production. In support of this, we found that Didox reduced FcεRI-mediated AP-1 and NFκB transcriptional activity. Finally, Didox suppressed mast cell-dependent, IgE-mediated passive systemic anaphylaxis in vivo. These data demonstrate the potential use for Didox asa means of antagonizing mast cell responses in allergic disease.

Alkannin inhibits CCL3 and CCL5 production in human periodontal ligament cells.

Alkannin, which is found in Alkanna tinctoria, a member of the borage family, is used as a food coloring. Alkannin has recently been reported to have certain biological functions, such as anti-microbial and anti-oxidant effects. It is known that CC chemokine receptor (CCR) 5-positive leukocytes contribute to alveolar bone resorption in periodontal lesions. The aim of this study was to examine whether alkannin inhibits the production of CC chemokine ligand (CCL) 3 and CCL5, which are CCR5 ligands, in human periodontal ligament cells (HPDLC). Interleukin (IL)-1ß induced CCL3 and CCL5 production in HPDLC. Alkannin inhibited IL-1ß-mediated CCL3 and CCL5 production in HPDLC in a dose-dependent manner. Moreover, we revealed that alkannin suppressed inhibitor of kappa B-α degradation in IL-1ß-stimulated HPDLC. In addition, a nuclear factor (NF)-κB inhibitor significantly inhibited CCL3 and CCL5 production in IL-1ß-stimulated HPDLC. These results demonstrate that alkannin inhibits CCR5 ligand production in IL-1ß-stimulated HPDLC by attenuating the NF-κB signaling pathway.

IL-5 triggers a cooperative cytokine network that promotes eosinophil precursor maturation.

Eosinophils originate in the bone marrow from an eosinophil lineage-committed, IL-5Rα-positive, hematopoietic progenitor (eosinophil progenitor). Indeed, IL-5 is recognized as a critical regulator of eosinophilia and has effects on eosinophil progenitors, eosinophil precursors, and mature eosinophils. However, substantial levels of eosinophils remain after IL-5 neutralization or genetic deletion, suggesting that there are alternative pathways for promoting eosinophilia. In this study, we investigated the contributory role of IL-5 accessory cytokines on the final stages of eosinophil differentiation. IL-5 stimulation of low-density bone marrow cells resulted in expression of a panel of cytokines and cytokine receptors, including several ligand-receptor pairs. Notably, IL-4 and IL-4Rα were expressed by eosinophil precursors and mature eosinophils. Signaling through IL-4Rα promoted eosinophil maturation when IL-5 was present, but IL-4 stimulation in the absence of IL-5 resulted in impaired eosinophil survival, suggesting that IL-4 cooperates with IL-5 to promote eosinophil differentiation. In contrast, CCL3, an eosinophil precursor-produced chemokine that signals through CCR1, promotes terminal differentiation of CCR1-positive eosinophil precursors in the absence of IL-5, highlighting an autocrine loop capable of sustaining eosinophil differentiation. These findings suggest that brief exposure to IL-5 is sufficient to initiate a cytokine cooperative network that promotes eosinophil differentiation of low-density bone marrow cells independent of further IL-5 stimulation.

CCL3 production by microglial cells modulates disease severity in murine models of retinal degeneration.

Many degenerative retinal diseases illustrate retinal inflammatory changes that include infiltration of microglia and macrophages into the subretinal space. In this study, we examined the role of chemokines in the Abca4(-/-)Rdh8(-/-) mouse model of Stargardt disease and the Mertk(-/-) mouse model of retinitis pigmentosa. PCR array analysis of 84 chemokines and related molecules revealed 84.6-fold elevated expression of Ccl3 (MIP-1a) 24 h after light exposure in Abca4(-/-)Rdh8(-/-) mice. Only MIP-1 chemokines, including Ccl3 and Ccl4, displayed peak expression 24 h after light exposure, and peaked earlier than the other chemokines. Secretion of Ccl3 was documented only in microglia, whereas both microglia and retinal pigment epithelium cells produced Ccl2. Exposure of Cx3Cr1(gfp/Δ)Abca4(-/-)Rdh8(-/-) mice to intense light resulted in the appearance of Cx3Cr1GFP(+) monocytes in the subretinal space. To address the in vivo role of CCL3 in retinal degeneration, Ccl3(-/-)Abca4(-/-)Rdh8(-/-) mice and Ccl3(-/-)Mertk(-/-) mice were generated. Following intense light exposure, Ccl3(-/-)Abca4(-/-)Rdh8(-/-) mice displayed persistent retinal inflammation with appearance of Iba-1(+) cells in the subretinal space, severe photoreceptor cell death, and increased Ccl4 expression compared with Abca4(-/-)Rdh8(-/-) mice. In contrast, Ccl3(-/-)Abca4(-/-)Rdh8(-/-) mice exhibited a milder retinal inflammation and degeneration than Abca4(-/-)Rdh8(-/-) mice did in age-related chronic retinal degeneration under room light conditions. The deficiency of Ccl3 also attenuated the severity of retinal degeneration in Mertk(-/-) mice. Taken together, our results indicate that Ccl3 has an essential role in regulating the severity of retinal inflammation and degeneration in these mouse models.

Cleavage of the T cell protein tyrosine phosphatase by the hepatitis C virus nonstructural 3/4A protease induces a Th1 to Th2 shift reversible by ribavirin therapy.

Ribavirin has proven to be a key component of hepatitis C therapies both involving IFNs and new direct-acting antivirals. The hepatitis C virus-mediated interference with intrahepatic immunity by cleavage of mitochondrial antiviral signaling protein (MAVS) and T cell protein tyrosine phosphatase (TCPTP) suggests an avenue for compounds that may counteract these effects. We therefore studied the effects of ribavirin, with or without inhibition of the nonstructural (NS)3/4A protease, on intrahepatic immunity. The intrahepatic immunity of wild-type and NS3/4A-transgenic mice was determined by Western blot, ELISA, flow cytometry, and survival analysis. Various MAVS or TCPTP constructs were injected hydrodynamically to study their relevance. Ribavirin pretreatment was performed in mice expressing a functional or inhibited NS3/4A protease to analyze its effect on NS3/4A-mediated changes. Intrahepatic NS3/4A expression made mice resistant to TNF-α-induced liver damage and caused an alteration of the intrahepatic cytokine (IFN-γ and IL-10) and chemokine (CCL3, CCL17, CCL22, CXCL9, and CXCL11) profiles toward an anti-inflammatory state. Consistent with this, the number of intrahepatic Th1 cells and IFN-γ(+) T cells in NS3/4A-transgenic mice decreased, whereas the amount of Th2 cells increased. These effects could be reversed by injection of uncleavable TCPTP but not uncleavable MAVS and were absent in a mouse expressing a nonfunctional NS3/4A protease. Importantly, the NS3/4A-mediated effects were reversed by ribavirin treatment. Thus, cleavage of TCPTP by NS3/4A induces a shift of the intrahepatic immune response toward a nonantiviral Th2-dominated immunity. These effects are reversed by ribavirin, supporting that ribavirin complements the effects of direct-acting antivirals as an immunomodulatory compound.

CCR1 inhibition ameliorates the progression of lupus nephritis in NZB/W mice.

Systemic lupus erythematosus is a chronic inflammatory autoimmune disease, the development of which is characterized by a progressive loss of renal function. Such dysfunction is associated with leukocyte infiltration in the glomerular and tubulointerstitial compartments in both human and experimental lupus nephritis. In this study, we investigated the role of the Ccr1 chemokine receptor in this infiltration process during the progression of nephritis in the lupus-prone New Zealand Black/New Zealand White (NZB/W) mouse model. We found that peripheral T cells, mononuclear phagocytes, and neutrophils, but not B cells, from nephritic NZB/W mice were more responsive to Ccr1 ligands than the leukocytes from younger prenephritic NZB/W mice. Short-term treatment of nephritic NZB/W mice with the orally available Ccr1 antagonist BL5923 decreased renal infiltration by T cells and macrophages. Longer Ccr1 blockade decreased kidney accumulation of effector/memory CD4(+) T cells, Ly6C(+) monocytes, and both M1 and M2 macrophages; reduced tubulointerstitial and glomerular injuries; delayed fatal proteinuria; and prolonged animal lifespan. In contrast, renal humoral immunity was unaffected in BL5923-treated mice, which reflected the unchanged numbers of infiltrated B cells in the kidneys. Altogether, these findings define a pivotal role for Ccr1 in the recruitment of T and mononuclear phagocyte cells to inflamed kidneys of NZB/W mice, which in turn contribute to the progression of renal injury.

IRGM3 contributes to immunopathology and is required for differentiation of antigen-specific effector CD8+ T cells in experimental cerebral malaria.

Gamma interferon (IFN-γ) drives antiparasite responses and immunopathology during infection with Plasmodium species. Immunity-related GTPases (IRGs) are a class of IFN-γ-dependent proteins that are essential for cell autonomous immunity to numerous intracellular pathogens. However, it is currently unknown whether IRGs modulate responses during malaria. We have used the Plasmodium berghei ANKA (PbA) model in which mice develop experimental cerebral malaria (ECM) to study the roles of IRGM1 and IRGM3 in immunopathology. Induction of mRNA for Irgm1 and Irgm3 was found in the brains and spleens of infected mice at times of peak IFN-γ production. Irgm3-/- but not Irgm1-/- mice were completely protected from the development of ECM, and this protection was associated with the decreased induction of inflammatory cytokines, as well as decreased recruitment and activation of CD8+ T cells within the brain. Although antigen-specific proliferation of transferred CD8+ T cells was not diminished compared to that of wild-type recipients following PbA infection, T cells transferred into Irgm3-/- recipients showed a striking impairment of effector differentiation. Decreased induction of several inflammatory cytokines and chemokines (interleukin-6, CCL2, CCL3, and CCL4), as well as enhanced mRNA expression of type-I IFNs, was found in the spleens of Irgm3-/- mice at day 4 postinfection. Together, these data suggest that protection from ECM pathology in Irgm3-/- mice occurs due to impaired generation of CD8+ effector function. This defect is nonintrinsic to CD8+ T cells. Instead, diminished T cell responses most likely result from defective initiation of inflammatory responses in myeloid cells.

APOE genotype-dependent modulation of astrocyte chemokine CCL3 production.

Apolipoprotein E (apoE) is well known as a regulator of cholesterol homeostasis, and is increasingly recognized to play a prominent role in the modulation of innate immune response, including cell-to-cell communication and migration. Alzheimer's disease (AD) is a slowly progressive neurodegenerative disorder characterized by neuroinflammation that appears to be an important component of the pathophysiology of the disease. Astrocytes are the majority cell type in brain, exerting significant influence over a range of central nervous system activities, including microglial-mediated neuroinflammatory responses. As the resident innate immune effector cells of the brain, microglia respond to soluble chemical signals released from tissue during injury and disease by mobilizing to lesion sites, clearing toxic molecules, and releasing chemical signals of their own. While microglial-mediated neuroinflammation in the AD brain remains an area of intense investigation, the mechanisms underlying reinforcement and regulation of these aberrant microglial responses by astrocytes are largely unstudied. Moreover, although inheritance of APOE ɛ4 represents the greatest genetic risk factor for sporadic AD, the mechanism by which apoE isoforms differentially influence AD pathophysiology is unknown. Here we show that APOE ɛ4 genotype specifically modulates astrocyte secretion of potent microglial chemotactic agents, including CCL3, thus providing evidence that APOE modulation of central nervous system (CNS) innate immune response is mediated through astrocytes.

In vitro progesterone modulation on bacterial endotoxin-induced production of IL-1ß, TNFα, IL-6, IL-8, IL-10, MIP-1α, and MMP-9 in pre-labor human term placenta.

BACKGROUND: During human pregnancy, infection/inflammation represents an important factor that increases the risk of developing preterm labor. The purpose of this study was to determine if pre-treatment with progesterone has an immunomodulatory effect on human placenta production of endotoxin-induced inflammation and degradation of extracellular matrix markers. METHODS: Placentas were obtained under sterile conditions from pregnancies delivered at term before the onset of labor by cesarean section. Explants from central cotyledons of 10 human placentas were pre-treated with different concentrations of progesterone (0.01, 01, 1.0 µM) and then stimulated with 1000 ng/mL of LPS of Escherichia coli. Cytokines TNFα, IL-1ß, IL-6, IL-8, MIP-1α, IL-10 concentrations in the culture medium were then measured by specific ELISA. Secretion profile of MMP-9 was evaluated by ELISA and zymogram. Statistical differences were determined by one-way ANOVA followed by the appropriate ad hoc test; P < 0.05 was considered statistically significant. RESULTS: In comparison to the explants incubated with vehicle, the LPS treatment led to a significant increase in the level of all cytokines. In comparison to the explants treated only with LPS, pre-treatment with 0.01-1.0 µM progesterone significantly blunted (73, 56, 56, 75, 25, 48 %) the secretion of TNF-α, IL-1ß, IL-6, IL-8, MIP-1α, IL-10, respectively. The MMP-9 induced by LPS treatment was inhibited only with the highest concentration of progesterone. Mifepristone (RU486) blocked the immunosuppressive effect of progesterone. CONCLUSIONS: The present results support the concept that progesterone could be part of the compensatory mechanism that limits the inflammation-induced cytotoxic effects associated with an infection process during gestation.

The histamine H4 receptor regulates chemokine production in human natural killer cells.

BACKGROUND: Natural killer (NK) cells have been detected in the lesional skin of patients with inflammatory skin diseases, where high levels of histamine are also present. Therefore, we investigated the effect of histamine, in particular via the histamine H4 receptor (H4R), on gene expression levels in human NK cells. METHODS: Comprehensive microarray-based mRNA expression profiling was performed to assess the gene expression levels in human NK cells in response to H4R stimulation in an unbiased approach. The expression of selected cytokines and chemokines was quantified by real-time PCR and enzyme-linked immunosorbent assay. RESULTS: The microarray analysis identified only few genes which were differentially regulated upon H4R stimulation. In follow-up studies, a significant upregulation of CCL3 and CCL4 at the mRNA level and in addition for CCL3 also at the protein level via the H4R was observed. CONCLUSION: The elevated expression levels of chemokines in response to H4R stimulation might foster the inflammation in allergic skin diseases and characterize the H4R as a promising therapeutic target.

Macrophage inflammatory protein-1 production and eosinophil infiltration in chronic rhinosinusitis with nasal polyps.

OBJECTIVE: Eosinophil recruitment to the nasal mucosa involves a number of chemokines. The aim of this study was to evaluate nasal secretion levels of macrophage inflammatory protein-1 alpha (MIP-1α) and MIP-1ß and to correlate these levels with clinical characteristics and degree of eosinophilia in nonallergic and allergic patients with nasal polyposis (NP). METHODS: Fourteen nonatopic and 14 atopic patients with NP were recruited for this cross-sectional study. Fourteen healthy subjects were included as controls. The concentrations of MIP-1α and MIP-1ß in nasal secretions were measured by flow cytometry. Eosinophil counts were performed by cytological examination of the scraped nasal mucosa. We scored each of the 28 patients according to the nasal symptom score, endoscopic score, and computed tomography (CT) score. RESULTS: We found significantly higher concentrations of MIP-1α in nasal fluid of nonallergic and allergic NP patients compared to control subjects. In nonallergic patients, we found positive correlations between MIP-1α levels and endoscopic score, CT score, and the percentage of eosinophils. CONCLUSION: MIP-1α may play a role in eosinophil recruitment in NP. Our results suggest that the measurement of MIP-1α in nasal secretions could be useful in evaluating the degree of eosinophil inflammation and severity of disease in nonallergic patients.

Chemokine expression of CCL2, CCL3, CCL5 and CXCL10 during early inflammatory tendon healing precedes nerve regeneration: an immunohistochemical study in the rat.

PURPOSE: Chemokines are major promoters of repair and may regulate nerve ingrowth that is essential in tendon healing. The purpose of this study was to assess the temporal occurrence of different chemokines during Achilles tendon healing in relation to sensory nerve regeneration. Chemokine presence in tendon healing has not been studied previously. METHODS: Chemokine expression, nerve regeneration, angiogenesis and inflammatory cell occurrence during healing of Achilles tendon rupture in the rat were studied by immunohistochemistry and histology including semiquantitative assessment. Markers for chemokines (CCL5, CCL2, CCL3, CXCL10), nerves (PGP-9.5) and sensory neuropeptide substance P (SP) were analysed at different time points (1 day-16 weeks) post-rupture. RESULTS: In intact tendons (controls) immunoreactivity to all chemokines, PGP-9.5 and SP were confined to the tendon surroundings. After rupture, there was rapid increase in the tendon proper of the chemokines studied, all exhibiting their peak expression at week 1. Subsequently, at weeks 2-6, emerging inflammatory cells and maximum sprouting of PGP-/SP-positive nerves were observed close to newly formed blood vessels within the tendon proper, while chemokine expression already decreased. During weeks 6-8, PGP-/SP-positive nerves withdrew from the rupture site and relocated together with the chemokines in the surrounding tendon. CONCLUSIONS: Early chemokine expression in the healing tendon precedes ingrowth of new nerves, angiogenesis and emergence of inflammatory cells. The fine-tuned temporal and spatial appearance of chemokines suggests a chemoattractant role for inflammatory cell migration and possibly also a role in angiogenesis and neurogenesis. Chemokines may thus exhibit vital targets for biological modulation of tendon repair.

Neuronal and epithelial cell rescue resolves chronic systemic inflammation in the lipid storage disorder Niemann-Pick C.

Chronic systemic inflammation is thought to be a major contributor to metabolic and neurodegenerative diseases. Since inflammatory components are shared among different disorders, targeting inflammation is an attractive option for mitigating disease. To test the significance of inflammation in the lipid storage disorder (LSD) Niemann-Pick C (NPC), we deleted the macrophage inflammatory gene Mip1a/Ccl3 from NPC diseased mice. Deletion of Ccl3 had been reported to delay neuronal loss in Sandhoff LSD mice by inhibiting macrophage infiltration. For NPC mice, in contrast, deleting Ccl3 did not retard neurodegeneration and worsened the clinical outcome. Depletion of visceral tissue macrophages also did not alter central nervous system (CNS) pathology and instead increased liver injury, suggesting a limited macrophage infiltration response into the CNS and a beneficial role of macrophage activity in visceral tissue. Prevention of neuron loss or liver injury, even at late stages in the disease, was achieved through specific rescue of NPC disease in neurons or in liver epithelial cells, respectively. Local epithelial cell correction was also sufficient to reduce the macrophage-associated pathology in lung tissue. These results demonstrate that elevated inflammation and macrophage activity does not necessarily contribute to neurodegeneration and tissue injury, and LSD defects in immune cells may not preclude an appropriate inflammatory response. We conclude that inflammation remains secondary to neuronal and epithelial cell dysfunction and does not irreversibly contribute to the pathogenic cascade in NPC disease. Without further exploration of possible beneficial roles of inflammatory mediators, targeting inflammation may not be therapeutically effective at ameliorating disease severity.

Allograft inflammatory factor-1 stimulates chemokine production and induces chemotaxis in human peripheral blood mononuclear cells.

Allograft inflammatory factor-1 (AIF-1) is expressed by macrophages, fibroblasts, endothelial cells and smooth muscle cells in immune-inflammatory disorders such as systemic sclerosis, rheumatoid arthritis and several vasculopathies. However, its molecular function is not fully understood. In this study, we examined gene expression profiles and induction of chemokines in monocytes treated with recombinant human AIF (rhAIF-1). Using the high-density oligonucleotide microarray technique, we compared mRNA expression profiles of rhAIF-1-stimulated CD14(+) peripheral blood mononuclear cells (CD14(+) PBMCs) derived from healthy volunteers. We demonstrated upregulation of genes for several CC chemokines such as CCL1, CCL2, CCL3, CCL7, and CCL20. Next, using ELISAs, we confirmed that rhAIF-1 promoted the secretion of CCL3/MIP-1α and IL-6 by CD14(+) PBMCs, whereas only small amounts of CCL1, CCL2/MCP-1, CCL7/MCP-3 and CCL20/MIP-3α were secreted. Conditioned media from rhAIF-1stimulated CD14(+) PBMCs resulted in migration of PBMCs. These findings suggest that AIF-1, which induced chemokines and enhanced chemotaxis of monocytes, may represent a molecular target for the therapy of immune-inflammatory disorders.

Autocrine CCL3 and CCL4 induced by the oncoprotein LMP1 promote Epstein-Barr virus-triggered B cell proliferation.

Epstein-Barr virus (EBV) alters the regulation and expression of a variety of cytokines in its host cells to modulate host immune surveillance and facilitate viral persistence. Using cytokine antibody arrays, we found that, in addition to the cytokines reported previously, two chemotactic cytokines, CCL3 and CCL4, were induced in EBV-infected B cells and were expressed at high levels in all EBV-immortalized lymphoblastoid cell lines (LCLs). Furthermore, EBV latent membrane protein 1 (LMP1)-mediated Jun N-terminal protein kinase activation was responsible for upregulation of CCL3 and CCL4. Inhibition of CCL3 and CCL4 in LCLs using a short hairpin RNA approach or by neutralizing antibodies suppressed cell proliferation and caused apoptosis, indicating that autocrine CCL3 and CCL4 are required for LCL survival and growth. Importantly, significant amounts of CCL3 were detected in EBV-positive plasma from immunocompromised patients, suggesting that EBV modulates this chemokine in vivo. This study reveals the regulatory mechanism and a novel function of CCL3 and CCL4 in EBV-infected B cells. CCL3 might be useful as a therapeutic target in EBV-associated lymphoproliferative diseases and malignancies.