MCP-1 In Response Research Articles

Screening and identification of host signaling pathways for alleviating influenza-induced production of pro-inflammatory cytokines, IP-10, IL-8, and MCP-1, using a U937 cell-based influenza model

Influenza virus infection initiates an exaggerated inflammatory response, which may culminate in a fatal cytokine storm characterized by the excessive production of pro-inflammatory cytokines. Prior research indicates that IP-10, IL-8, and MCP-1, primarily produced by monocytes and macrophages, play a crucial role in influenza-induced inflammation. The lung injury from influenza virus infection can be mitigated by suppressing or inhibiting these cytokines through knockout, knockdown, or targeted intervention approaches. To identify the key host signaling pathways responsible for producing pro-inflammatory cytokines, we utilized a U937 cell model that secretes IP-10, IL-8, and MCP-1 in response to influenza infection. This model has been previously validated in our laboratory as an appropriate system for screening anti-inflammatory agents and potential drug targets. We conducted a screening assay employing an inhibitor library consisting of 2,138 compounds that target various known pathways and host factors. Our findings indicated that inhibitors targeting protein tyrosine kinases and mitogen-activated protein kinases demonstrated superior efficacy in suppressing cytokine production induced by influenza A virus infection compared to inhibitors aimed at other host factors. Notably, a substantial proportion of the identified hits capable of inhibiting the expression of all three cytokines in the secondary screening were classified as tyrosine kinase inhibitors. Validation experiments further established that Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathways, along with p38 MAPK and Raf–MEK–ERK pathways, are the principal regulators of pro-inflammatory cytokine expression in monocytes and macrophages. Moreover, our results suggest that TKIs present promising opportunities as novel therapeutic agents against influenza-induced pneumonia.

TLR2 Regulates Mast Cell IL-6 and IL-13 Production During Listeria monocytogenes Infection.

Listeria monocytogenes (L.m) is efficiently controlled by several cells of the innate immunity, including the Mast Cell (MC). MC is activated by L.m inducing its degranulation, cytokine production and microbicidal mechanisms. TLR2 is required for the optimal control of L.m infection by different cells of the immune system. However, little is known about the MC receptors involved in recognizing this bacterium and whether these interactions mediate MC activation. In this study, we analyzed whether TLR2 is involved in mediating different MC activation responses during L.m infection. We found that despite MC were infected with L.m, they were able to clear the bacterial load. In addition, MC degranulated and produced ROS, TNF-α, IL-1β, IL-6, IL-13 and MCP-1 in response to bacterial infection. Interestingly, L.m induced the activation of signaling proteins: ERK, p38 and NF-κB. When TLR2 was blocked, L.m endocytosis, bactericidal activity, ROS production and mast cell degranulation were not affected. Interestingly, only IL-6 and IL-13 production were affected when TLR2 was inhibited in response to L.m infection. Furthermore, p38 activation depended on TLR2, but not ERK or NF-κB activation. These results indicate that TLR2 mediates only some MC activation pathways during L.m infection, mainly those related to IL-6 and IL-13 production.

Spred2-deficiency enhances the proliferation of lung epithelial cells and alleviates pulmonary fibrosis induced by bleomycin

The mitogen-activated protein kinase (MAPK) pathways are involved in many cellular processes, including the development of fibrosis. Here, we examined the role of Sprouty-related EVH-1-domain-containing protein (Spred) 2, a negative regulator of the MAPK-ERK pathway, in the development of bleomycin (BLM)-induced pulmonary fibrosis (PF). Compared to WT mice, Spred2−/− mice developed milder PF with increased proliferation of bronchial epithelial cells. Spred2−/− lung epithelial cells or MLE-12 cells treated with spred2 siRNA proliferated faster than control cells in vitro. Spred2−/− and WT macrophages produced similar levels of TNFα and MCP-1 in response to BLM or lipopolysaccharide and myeloid cell-specific deletion of Spred2 in mice had no effect. Spred2−/− fibroblasts proliferated faster and produced similar levels of MCP-1 compared to WT fibroblasts. Spred2 mRNA was almost exclusively detected in bronchial epithelial cells of naïve WT mice and it accumulated in approximately 50% of cells with a characteristic of Clara cells, 14 days after BLM treatment. These results suggest that Spred2 is involved in the regulation of tissue repair after BLM-induced lung injury and increased proliferation of lung bronchial cells in Spred2−/− mice may contribute to faster tissue repair. Thus, Spred2 may present a new therapeutic target for the treatment of PF.

OxLDL-mediated immunologic memory in endothelial cells

Trained innate immunity describes the metabolic reprogramming and long-term proinflammatory activation of innate immune cells in response to different pathogen or damage associated molecular patterns, such as oxidized low-density lipoprotein (oxLDL). Here, we have investigated whether the regulatory networks of trained innate immunity also control endothelial cell activation following oxLDL treatment. Human aortic endothelial cells (HAECs) were primed with oxLDL for 24 h. After a resting time of 4 days, cells were restimulated with the TLR2-agonist PAM3cys4. OxLDL priming induced a proinflammatory memory with increased production of inflammatory cytokines such as IL-6, IL-8 and MCP-1 in response to PAM3cys4 restimulation. This memory formation was dependent on TLR2 activation. Furthermore, oxLDL priming of HAECs caused characteristic metabolic and epigenetic reprogramming, including activation of mTOR-HIF1α-signaling with increases in glucose consumption and lactate production, as well as epigenetic modifications in inflammatory gene promoters. Inhibition of mTOR-HIF1α-signaling or histone methyltransferases blocked the observed phenotype. Furthermore, primed HAECs showed epigenetic activation of ICAM-1 and increased ICAM-1 expression in a HIF1α-dependent manner. Accordingly, live cell imaging revealed increased monocyte adhesion and transmigration following oxLDL priming. In summary, we demonstrate that oxLDL-mediated endothelial cell activation represents an immunologic event, which triggers metabolic and epigenetic reprogramming. Molecular mechanisms regulating trained innate immunity in innate immune cells also regulate this sustained proinflammatory phenotype in HAECs with enhanced atheroprone cell functions. Further research is necessary to elucidate the detailed metabolic regulation and the functional relevance for atherosclerosis formation in vivo.

Effect of implant surface material and roughness to the susceptibility of primary gingival fibroblasts to inflammatory stimuli

ObjectivesThe impact of the implant surface material and roughness on inflammatory processes in peri-implantitis is not entirely clear. Hence, we investigated how titanium and zirconia surfaces with different roughness influence the susceptibility of primary human gingival fibroblasts to different inflammatory stimuli. MethodsPrimary human gingival fibroblasts were isolated from 8 healthy individuals and cultured on following surfaces: smooth titanium machined surface (TiM), smooth zirconia machined surface (ZrM), moderately rough titanium surface (SLA), or moderately rough zirconia surface (ZLA). Subsequently, stimulation with one of the following stimuli was performed: Porphyromonas gingivalis lipopolysaccharide (LPS), tumor necrosis factor (TNF)-α, interleukin (IL)-1β. The resulting production of IL-6, IL-8, and monocyte chemoattractant protein (MCP)-1 was measured by qPCR and ELISA. ResultsP. gingivalis LPS induced IL-6 and MCP-1 production was slightly higher on titanium surfaces compared to zirconia surfaces. IL-1β induced IL-6 production was not affected by any surface characteristic. The production of MCP-1 in response to IL-1β was higher on smooth compared to rough surfaces and was not affected by the material. The production of IL-6 and MCP-1 in response to TNF-α was most strongly affected by surface characteristics. Higher production of these cytokine was observed on smooth compared to rough surfaces and on titanium compared to zirconia surfaces. Surface characteristics had only minor effects on IL-8 production. SignificanceThe susceptibility of primary gingival fibroblasts to inflammation depends on various factors, such as surface material, surface roughness and the nature of inflammatory stimuli. All these factors might determine susceptibility to peri-implantitis.

1532. Increased Risk of Bacterial, Fungal and Other Viral Infections During CMV Infection: Decreased Cytokine Production in Response to Toll-Like Receptor Ligands

BackgroundIn the solid-organ transplant (SOT) setting, CMV is an immunomodulatory virus that indirectly increases the risk for bacterial, fungal and viral infections. However, the pathogenesis of this phenomenon is poorly understood. The aim of our study was to determine whether inflammatory responses to different Toll-like receptor ligands are blunted during CMV infection in SOT patients.MethodsCMV D+/R− SOT patients had blood drawn at the end of CMV prophylaxis and then weekly after onset of CMV viremia. PBMCs were extracted and incubated for 24 hours in the presence of bacterial (LPS), fungal (Zymosan [ZYM]), and viral (Resiquimod [R848]) ligands. Proinflammatory (IL1β), Th1 (IFNγ), Th2 (IL4), immunoregulatory (IL10), and chemotactic (MCP1) cytokines were measured in the supernatant by multiplex ELISA.ResultsThirty-eight SOT patients were followed for at least 9 months. Patients who developed subsequent CMV infection had lower cytokines in response to bacterial, fungal and viral ligands (LPS, ZYM, and R848) at the end of prophylaxis compared with those with no CMV infection. These results were independent of immunosuppression and peripheral blood cell counts. Specifically, these trends were significantly different with respect to IFNγ, IL1β, and IL10 production in response to LPS (P = 0.003, 0.003, and 0.039, respectively), R848 (P < 0.001, 0.039, and <0.001, respectively) and ZYM (P = 0.039, 0.003, and 0.003, respectively), as well as for MCP1 in response to R848 or ZYM (P = 0.039 for both). In the cohort with CMV infection, cytokine responses to TLR ligands were even lower during the acute CMV infection when compared with the end of prophylaxis, although this was significant only for IL10 production after R848 stimulation (P = 0.034). There was no influence of CMV viral load or duration of viremia on cytokine levels.ConclusionResponse to non-CMV antigens during CMV infection was blunted supporting the clinical observation in transplant recipients that CMV infection increases susceptibility to bacterial, fungal, and other viral infections. However, inherent differences in patients that are neither directly related to CMV nor to their net level of immunosuppression also contribute to this increased susceptibility, as cytokine levels at the end of prophylaxis were lower among patients with compared with those without subsequent CMV infection.Disclosures All authors: No reported disclosures.

Pleiotrophin regulates microglia-mediated neuroinflammation

BackgroundPleiotrophin (PTN) is a cytokine found highly upregulated in the brain in different disorders characterized by overt neuroinflammation such as neurodegenerative diseases, drug addiction, traumatic injury, and ischemia. In the present work, we have explored whether PTN modulates neuroinflammation and if Toll-like receptor 4 (TLR4), crucial in the initiation of an immune response, is involved.MethodsIn immunohistochemistry assays, we studied lipopolysaccharide (LPS, 7.5 mg/kg i.p.)-induced changes in glial fibrillary acidic protein (GFAP, astrocyte marker) and ionized calcium-binding adaptor molecule 1 (Iba1, microglia marker) expression in the prefrontal cortex (PFC) and striatum of mice with transgenic PTN overexpression in the brain (PTN-Tg) and in wild-type (WT) mice. Cytokine protein levels were assessed in the PFC by X-MAP technology. The influence of TLR4 signaling in LPS effects in both genotypes was assessed by pretreatment with the TLR4 antagonist (TAK-242, 3.0 mg/kg i.p.). Murine BV2 microglial cells were treated with PTN (0.5 μg/ml) and LPS (1.0 μg/ml) and assessed for the release of nitric oxide (NO).ResultsWe found that LPS-induced microglial activation is significantly increased in the PFC of PTN-Tg mice compared to that of WT mice. The levels of TNF-α, IL-6, and MCP-1 in response to LPS were significantly increased in the PFC of PTN-Tg mice compared to that of WT mice. Pretreatment with TAK-242 efficiently blocked increases in cytokine contents in a similar manner in both genotypes. Concomitant incubation of BV2 cells with LPS and PTN significantly potentiated the production of NO compared to cells only treated with LPS.ConclusionsOur findings identify for the first time that PTN is a novel and potent regulator of neuroinflammation. Pleiotrophin potentiates LPS-stimulated microglia activation. Our results suggest that regulation of the PTN signaling pathways may constitute new therapeutic opportunities particularly in those neurological disorders characterized by increased PTN cerebral levels and neuroinflammation.

Abstract 570: Deficiency of Inhibitory Fcgamma Receptor II Does Not Exacerbate Atherosclerosis

Objective: Functionally, Fcgamma receptors (FcgRs) can be classified as activating (FcgRI, III, and IV) and inhibitory (FcgRII, CD32b) receptors. We have reported that deletion of activating FcgRs in apoE knockout mice decreased atherosclerosis. In this report we investigated the hypothesis that the deficiency of inhibitory CD32b exacerbates atherosclerosis in a hypercholesterolemic mouse model. Approach and Results: ApoE-CD32b double knockout mice congenic to the C57BL/6 (apoE-CD32b B6 -/-) were generated and atherosclerotic lesions were assessed. Contrary to our hypothesis, arterial lesions were significantly decreased in chow or high fat diet fed apoE-CD32b B6 -/- male and female mice, relative to apoE-/- mice . Bone marrow chimera approach using apoE-/- mice transplanted with apoE-CD32b B6 -/- marrow also showed significantly reduced arterial lesions. ApoE-CD32b B6 -/- mice had increased levels of IL-10 and TGF-b by CD4+ T cells, while IFN-g and IL-17 was decreased. As our findings conflict with a previous report using apoE-CD32b 129/6 -/- mixed background, we investigated if strain differences contributed to the anti-inflammatory response. Macrophages from mixed apoE-CD32b 129/B6 -/- mice showed more IL-1b, IL-6 and MCP-1 in response to immune complexes, while congenic CD32b B6 -/- mice showed more IL-10 and significantly reduced IL-1b. Interestingly expression of lupus-associated slam genes, located in close proximity to cd32b in mouse chromosome 1, is upregulated only in mixed CD32b 129/B6 -/- mice. Conclusions: Our findings demonstrate a detrimental role for CD32b signaling in atherosclerosis and the contribution of anti-inflammatory cytokine responses in the attenuated lesions observed in apoE-CD32b B6 -/- mice. As 129/sv genome derived lupus associated genes have been implicated in lupus phenotype in CD32b 129/B6 -/- mice our findings suggest possible epistatic mechanism contributing to the decreased lesions.

Effect of elevated carbon dioxide on bronchial epithelial innate immune receptor response to organic dust from swine confinement barns.

Hypercapnia is known to have immunoregulatory effects within the lung. Cell culture systems demonstrate this in both macrophages and alveolar cell lines, suggesting that the alveoli are affected by changes in CO2 levels. We hypothesized that hypercapnia would also modulate human bronchial epithelial cell immune responses. Innate immune responses to Pam3CSK4 (TLR2 ligand), LPS (TLR4 ligand) and a complex innate immune stimulus, an extract from the organic dust of swine confinement barns (barn dust extract or BDE), were tested in a human bronchial epithelial cell line, BEAS-2B. Both TLR ligands showed a decrease in IL-6 and IL-8 production, and an increase in MCP-1 in response to elevated CO2 indicating an enhancement in cytokine production to hypercapnia. This change was not reflected in expression levels of TLR receptor RNA which remained unchanged in response to elevated CO2. Interestingly, barn dust showed an increase in IL-6, IL-8 and MCP-1 response at 9% CO2, suggesting that elevated CO2 exerts different effects on different stimuli. Our results show that airway epithelial cell immune responses to barn dust respond differently to hypercapnic conditions than individual TLR ligands.

Recruited alveolar macrophages, in response to airway epithelial-derived monocyte chemoattractant protein 1/CCl2, regulate airway inflammation and remodeling in allergic asthma.

Although alveolar macrophages (AMs) from patients with asthma are known to be functionally different from those of healthy individuals, the mechanism by which this transformation occurs has not been fully elucidated in asthma. The goal of this study was to define the mechanisms that control AM phenotypic and functional transformation in response to acute allergic airway inflammation. The phenotype and functional characteristics of AMs obtained from human subjects with asthma after subsegmental bronchoprovocation with allergen was studied. Using macrophage-depleted mice, the role and trafficking of AM populations was determined using an acute allergic lung inflammation model. We observed that depletion of AMs in a mouse allergic asthma model attenuates Th2-type allergic lung inflammation and its consequent airway remodeling. In both human and mouse, endobronchial challenge with allergen induced a marked increase in monocyte chemotactic proteins (MCPs) in bronchoalveolar fluid, concomitant with the rapid appearance of a monocyte-derived population of AMs. Furthermore, airway allergen challenge of allergic subjects with mild asthma skewed the pattern of AM gene expression toward high levels of the receptor for MCP1 (CCR2/MCP1R) and expression of M2 phenotypic proteins, whereas most proinflammatory genes were highly suppressed. CCL2/MCP-1 gene expression was prominent in bronchial epithelial cells in a mouse allergic asthma model, and in vitro studies indicate that bronchial epithelial cells produced abundant MCP-1 in response to house dust mite allergen. Thus, our study indicates that bronchial allergen challenge induces the recruitment of blood monocytes along a chemotactic gradient generated by allergen-exposed bronchial epithelial cells.

Characterization of endocannabinoid-mediated induction of myeloid-derived suppressor cells involving mast cells and MCP-1

Endocannabinoids are lipid-signaling molecules found in the nervous system; however, their precise role in the periphery is unclear. In the current study, we observed that a single i.p. administration of AEA caused rapid induction of MDSCs. The MDSCs contained a mixture of granulocytic and monocytic subtypes and expressed Arg-1 and iNOS. The MDSCs suppressed T cell proliferation in vitro and used iNOS to mediate their effect. Moreover, adoptive transfer of MDSCs led to suppression of mBSA-induced DTH. Through the use of pharmacological inhibition, as well as genetic knockout mice, we found that the induction of MDSCs by AEA was CB1-dependent. The induction of MDSCs by AEA was reduced significantly in mast cell-deficient mice, while maintained in LPS-insensitive mice, showing that the induction of MDSCs by AEA was dependent, at least in part, on mast cells and independent of TLR4. Chemokine analysis of AEA- treated WT mice showed an early spike of MCP-1, which was decreased in Kit(W/W-sh) mice, showing a role of mast cells in the secretion of MCP-1 in response to AEA. Also, use of antibodies against MCP-1 or mice deficient in MCP-1 confirmed the role played by MCP-1. Interestingly, MCP-1 played a significant role in the induction of monocytic but not granulocytic MDSCs. Our studies demonstrate for the first time that endocannaboinids activate CB1 on mast cells to induce MCP-1, which facilitates recruitment of monocytic MDSCs.

Nitro-oleic acid protects against adriamycin-induced nephropathy in mice

Adriamycin (ADR) administration in susceptible rodents such as the BALB/c mouse strain produces injury to the glomerulus mimicking human focal glomerular sclerosis. The goal of the present study was to use this model to investigate antiproteinuric action of nitro-oleic acid (OA-NO2), a nitric oxide-derived endogenous lipid product, which has exhibited multiple attractive signaling properties particularly in the kidney. BALB/c mice were pretreated for 2 days with OA-NO2 at 5 mg·kg(-1)·day(-1) via an osmotic minipump, followed by a single injection of vehicle or adriamycin (10 mg/kg) via the tail vein. Albuminuria and renal function were analyzed at 1 wk post-ADR treatment. ADR mice developed prominent albuminuria, hypoalbuminemia, hyperlipidemia, and severe ascites. In contrast, the symptoms of nephrotic syndrome were greatly improved by OA-NO2 treatment. In parallel, plasma creatinine and plasma urea nitrogen were elevated in the ADR group, and the severity was less in the ADR+OA-NO2 group. OA-NO2 attenuates ADR-induced glomerulosclerosis, podocyte loss, and tubulointerstitial fibrosis. Indices of oxidative stress, including plasma and urinary thiobarbituric acid-reactive substances and renal expression of NAD(P)H oxidase p47(phox) and gp91(phox), and inflammation, including renal expression of TNF-α, IL-1β, and MCP-1 in response to ADR, were all similarly suppressed. Together, these findings suggest that OA-NO2 exerts renoprotective action against ADR nephropathy likely via its anti-inflammatory and antioxidant properties.

Bone marrow mesenchymal stem cells calibrate the innate immune tone by inducing monocyte emigration in response to circulating TLR-ligands (162.1)

Abstract Inflammatory (Ly6Chi CCR2+) monocytes provide defense against infections but also contribute to autoimmune diseases and atherosclerosis. Monocytes originate from the bone marrow and their entry into the bloodstream requires stimulation of CCR2 chemokine receptors by MCP1. How monocyte emigration from bone marrow is triggered by infections in anatomically remote sites remains unclear. We demonstrate that low concentrations of TLR ligands in the bloodstream drive CCR2-dependent emigration of monocytes from bone marrow. Bone marrow mesenchymal stem cells (MSCs) rapidly express MCP1 in response to circulating TLR ligands or systemic bacterial infection and induce inflammatory monocyte trafficking into the bloodstream. Targeted deletion of MCP1 from MSCs impaired monocyte emigration from bone marrow upon LPS stimulation and increased susceptibility to Listeria monocytogenes infection. Our findings suggest that bone marrow MSCs respond to circulating microbial molecules and regulate bloodstream monocyte frequencies by secreting MCP1 in proximity to bone marrow vascular sinuses.

Sphingosine-1-Phosphate Receptor 3 Promotes Recruitment of Monocyte/Macrophages in Inflammation and Atherosclerosis

The role of sphingosine-1-phosphate (S1P) and its receptors in the pathogenesis of atherosclerosis has not been investigated. We hypothesized that the S1P receptor 3 (S1P(3)) plays a causal role in the pathogenesis of atherosclerosis. We examined atherosclerotic lesion development in mice deficient for S1P(3) and apolipoprotein (Apo)E. Although S1P(3) deficiency did not affect lesion size after 25 or 45 weeks of normal chow diet, it resulted in a dramatic reduction of the monocyte/macrophage content in lesions of S1P(3)(-/-)/ApoE(-/-) double knockout mice. To search for putative defects in monocyte/macrophage recruitment, we examined macrophage-driven inflammation during thioglycollate-induced peritonitis. Elicited peritoneal macrophages were reduced in S1P(3)-deficient mice and expressed lower levels of tumor necrosis factor-α and monocyte chemoattractant protein-1. Bone marrow-derived S1P(3)-deficient macrophages produced less MCP-1 in response to lipopolysaccharide stimulation. In vitro, S1P was chemotactic for wild-type but not S1P(3)-deficient peritoneal macrophages. In vivo, S1P concentration increased rapidly in the peritoneal cavity after initiation of peritonitis. Treatment with the S1P analog FTY720 attenuated macrophage recruitment to the peritoneum. Studies in bone marrow chimeras showed that S1P(3) in both hematopoietic and nonhematopoietic cells contributed to monocyte/macrophage accumulation in atherosclerotic lesions. Finally, S1P(3) deficiency increased the smooth muscle cell content of atherosclerotic lesions and enhanced neointima formation after carotid ligation arguing for an antiproliferative/antimigratory role of S1P(3) in the arterial injury response. Our data suggest that S1P(3) mediates the chemotactic effect of S1P in macrophages in vitro and in vivo and plays a causal role in atherosclerosis by promoting inflammatory monocyte/macrophage recruitment and altering smooth muscle cell behavior.

Retraction: Endogenous IL-15 Sustains Recruitment of IL-2Rβ and Common γ and IL-2-Mediated Chemokine Production in Normal and Inflamed Human Gingival Fibroblasts

We recently reported that anti-IL-15 neutralizing mAb has been shown to inhibit production of MCP-1 in response to IL-2 from normal human gingival fibroblasts (HGF), the major constituent of gingival tissue. In the present study, we examined the expression of IL-2R and IL-15R subunits in HGF from normal and inflamed regions and the role of endogenous IL-15 in IL-2-mediated signaling. Normal HGF expressed IL-2Rβ and common γ-chain (γ c ) but not IL-2Rα or IL-15Rα, whereas inflamed HGF expressed IL-2Rα, IL-15Rα, IL-2Rβ, and γ c , as assessed by RT-PCR and flow cytometry. Exogenous IL-2 and IL-15 induced production of MCP-1 but not IL-8 in normal HGF, and induced the production of both chemokines in inflamed HGF. Both HGF constitutively transcribed the 48 aa-IL-15 isoform, and the isoform was not actively secreted but rather existed as a membrane-bound form. Pretreatment with anti-IL-15 neutralizing mAb for 24 h completely inhibited the production of MCP-1 induced by IL-2 and IL-15 and IL-2-induced phosphorylation of Jak 1 and 3 in HGF. The pretreatment and RNA interference targeted to IL-15 mRNA resulted in total inhibition of the IL-2Rβ and γ c expression at mRNA and protein levels. Furthermore, excess amounts of IL-2 restored the inhibitory effect of anti-IL-15, inhibition of NF-κB abrogated the expression of IL-2Rβ and γ c , and IL-2-induced-nuclear translocation of NF-κB was completely inhibited by the RNA interference in HGF. These results suggest that endogenous membrane-bound IL-15 sustains recruitment of IL-2Rβ and γ c through activation of NF-κB in HGF.

Macrophages express a TCRαβ-based variable immunoreceptor (134.34)

Abstract Immunological dogma holds that variable immunoreceptors are restricted to lymphocytes. However, recent studies demonstrate that a subpopuation of human neutrophils expresses the TCRαβ (PNAS 103:14441, 2006) and mouse thymic granulocytes exhibit rearrangement of their TCR loci (Nature 452:764, 2008). Here, we provide evidence that a subpopulation of circulating human monocytes, monocyte-derived macrophages and tissue macrophages constitutively express a TCRαβ-based variable immunoreceptor. TCRαβ + monocyte/macrophage subpopulations were assessed using with laser scanning cytometry and repertoire diversities were determined by CDR3 spectratyping. We find that human macrophages express individual-specific TCR Vαβ repertoires. Th1 or Th2 cytokine dependent in vitro differentiation triggers expression of distinct Vαβ repertoires in macrophages. Moreover, Th1 polarized macrophages show enhanced secretion of the major monocyte chemoattractant MCP-1 in response to costimulation with αCD3/CD28 . The presence of a variable immunoreceptor in macrophages together with our previous identification of the TCR in a subset of neutrophils challenge the traditional dichotomy of the vertebrate immune system into non-specific/myeloid and antigen-specific/lymphoid immunity. They unravel a much closer kinship between both lineages than commonly assumed. Supported by DFG grants KA 1078/4-1, BE 1768/5-1

Acquisition of Anergy to Proinflammatory Cytokines in Nonimmune Cells through Endoplasmic Reticulum Stress Response: A Mechanism for Subsidence of Inflammation

Acute endoplasmic reticulum (ER) stress causes induction of inflammatory molecules via activation of NF-kappaB. However, we found that, under ER stress conditions, renal mesangial cells acquire anergy to proinflammatory stimuli. Priming of the cells with ER stress inducers (tunicamycin, thapsigargin, A23187, and AB5 subtilase cytotoxin) caused blunted induction of MCP-1 in response to TNF-alpha, IL-1beta, macrophage-derived factors, or bystander macrophages. The magnitude of suppression was closely correlated with the level of GRP78, an endogenous indicator of ER stress. The suppression of MCP-1 under ER stress conditions was reversible and observed in general regardless of cell types or triggers of ER stress. The decrease in the level of MCP-1 mRNA was ascribed to transcriptional suppression via unexpected inhibition of NF-kappaB, but not to accelerated mRNA degradation. Subsequent experiments revealed that TNFR-associated factor 2, an essential component for TNF-alpha signaling, was down-regulated by ER stress. We also found that, under ER stress conditions, expression of NF-kappaB suppressor A20 was induced. Overexpression of A20 resulted in suppression of cytokine-triggered NF-kappaB activation and knockdown of A20 by RNA interference significantly attenuated induction of anergy by ER stress. In contrast, other ER stress-inducible/-related molecules that may suppress NF-kappaB (e.g., GRP78, NO, reactive oxygen species, and IkappaB) were not involved in the inhibitory effects of ER stress. These results elucidated ER stress-dependent mechanisms by which nonimmune cells acquire anergy to inflammatory stimuli under pathological situations. This self-defense machinery may play a role in halting progression of acute inflammation and in its spontaneous subsidence.

Amyloid-β Deposits Lead to Retinal Degeneration in a Mouse Model of Alzheimer Disease

To compare the temporal and spatial expression patterns of amyloid precursor protein (APP), amyloid-beta deposits, inflammatory chemokines, and apoptosis in the retina of a mouse model of Alzheimer disease (AD). Retinas of transgenic mice harboring a mutant presenilin (PS1) and a mutant APP gene were processed for TUNEL and immunohistochemistry with antibodies against APP, amyloid-beta, monocyte chemotactic protein (MCP)-1, and F4/80. Comparisons were made between age groups and between transgenic and wild-type congeners. The neuroretina demonstrated age-dependent increases in APP in the ganglion cells (RGCs) and in neurons of the inner nuclear layer (INL). Amyloid-beta demonstrated significant age-dependent deposition in the nerve fiber layer (NFL). TUNEL-positive RGC increased in an age-dependent fashion and in transgenic compared with wild-type congeners. Concomitant overexpression of MCP-1 and intense immunoreactivity for F4/80 suggested that RGCs upregulate MCP-1 in response to amyloid-beta. Activated microglia proliferated in response to MCP-1. In the outer retina, retinal pigment epithelium (RPE) demonstrated moderate age-dependent APP immunoreactivity, but nearby drusenlike deposits were not present. Amyloid-beta was observed in the choriocapillaris of the older animals. Amyloid-beta deposits accumulate with age in the retina of a transgenic mouse model of AD. The amyloid-beta loads are accompanied by increased immunoreactivity for MCP-1, F4/80, and TUNEL-positive profiles in the RGC layer. The results suggest that amyloid-beta causes neurodegeneration in the retina of the doubly mutant transgenic mouse model of AD.

Involvement of TWEAK/Fn14 interaction in the synovial inflammation of RA

TWEAK, TNF-like weak inducer of apoptosis, induces not only apoptosis of some tumour cells, but also proliferation of endothelial cells, and angiogenesis. It is known that TWEAK induces production of cytokines that are involved in the pathogenesis of RA. However, it is not clear how TWEAK takes part in the synovitis of RA. In this study, we investigated the role of TWEAK/fibroblast growth factor-inducible 14 (Fn14) interaction in the synovitis of RA. TWEAK and Fn14 expression on RA and OA synovial cells (SCs) were analysed by FACS. Synovial fibroblasts (SFs) or freshly isolated SCs were cultured in the presence or absence of recombinant TWEAK (rTWEAK) and anti-TWEAK or anti-Fn14 mAbs. Cell proliferation, cytokine/chemokine production and intercellular adhesion molecule (ICAM-1) expression were measured by WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium monosodium salt], ELISA and FACS, respectively. TWEAK expression was detected on CD45-positive population in RA synovium, whereas Fn14 was detected on both CD45-positive and CD45-negative populations. Cultured RA and OA SFs showed higher proliferation and produced IL-6, IL-8 and MCP-1 in response to rTWEAK. Cell proliferation and cytokine production of freshly isolated SCs from RA patients were suppressed by anti-TWEAK and anti-Fn14 mAbs. ICAM-1 expression on RA, but not OA, SFs was up-regulated by rTWEAK. These data suggest that TWEAK/Fn14 interaction plays a substantial role in the synovitis of RA, by directly inducing the proliferation of SFs, and by up-regulating the production of inflammatory cytokines/chemokines as well as the expression of ICAM-1.

AKAP13, a RhoA GTPase-specific Guanine Exchange Factor, Is a Novel Regulator of TLR2 Signaling

Members of the guanine exchange factor (GEF) family of scaffold proteins are involved in the integration of signal flow downstream of many receptors in adaptive immunity. However, the full complement of GEFs that function downstream of Toll-like receptors (TLRs) requires further identification and functional understanding. By systematically integrating expression profiles from immune and epithelial cells with functional studies, we demonstrate that protein kinase A anchoring protein 13 (AKAP13), a scaffold protein with GEF activity, is an activator of NF-kappaB downstream of TLR2 signaling. Stimulation of the human macrophage cell line THP-1 and epithelial cells with a TLR2 ligand caused a significant up-regulation in AKAP13 mRNA, corresponding to an increase in protein expression. Analysis of TLR2 reporter cell lines deficient in AKAP13 expression revealed significantly reduced NF-kappaB activation and reduced secretion of interleukin-8 and MCP-1 in response to specific ligand stimulation. Furthermore, NF-kappaB activation was partially inhibited by a GEF-deficient AKAP13 mutant. AKAP13 was also involved in phosphorylation of JNK but not of extracellular signal-regulated kinase ERK1 and -2 following ligand stimulation. Together, our results suggest that AKAP13 plays a role in TLR2-mediated NF-kappaB activation and suggest that GEF-containing scaffold proteins may confer specificity to innate immune responses downstream of TLRs.