Infiltration Of Mononuclear Phagocytes Research Articles

The role of the host-microbiome and metabolomics in sarcoidosis.

Sarcoidosis is a complex inflammatory fibrotic disease that affects multiple organ systems. It is characterised by the infiltration of lymphocytes and mononuclear phagocytes, which form non-caseating granulomas in affected organs. The lungs and intrathoracic lymph nodes are the most commonly affected organs. The underlying cause of sarcoidosis is unknown, but it is believed to occur in genetically predisposed individuals who are exposed to pathogenic organisms, environmental contaminants, or self and non-self-antigens. Recent research has suggested that the microbiome may play a role in the development of respiratory conditions, including sarcoidosis. Additionally, metabolomic studies have identified potential biomarkers for monitoring sarcoidosis progression. This review will focus on recent microbiome and metabolomic findings in sarcoidosis, with the goal of shedding light on the pathogenesis and possible diagnostic and therapeutic approaches.

Characterization of Cell Subsets Associated With Prognosis of Osteosarcoma Based on Single-Cell Sequencing Data

Objective To explore the cell subsets and characteristics related to the prognosis of osteosarcoma by analyzing the cellular composition of tumor tissue samples from different osteosarcoma patients.Methods The single-cell sequencing data and bulk sequencing data of different osteosarcoma patients were downloaded.We extracted the information of cell samples for dimensionality reduction,annotation,and cell function analysis,so as to identify the cell subsets and clarify the cell characteristics related to the prognosis of osteosarcoma.The development trajectory of macrophages with prognostic significance was analyzed,and the prognostic model of osteosarcoma was established based on the differentially expressed genes of macrophage differentiation.Results The cellular composition presented heterogeneity in the patients with osteosarcoma.The infiltration of mononuclear phagocytes in osteosarcoma had prognostic significance(P=0.003).Four macrophage subsets were associated with prognosis,and their signature transcription factors included RUNX3(+),ETS1(+),HOXD11(+),ZNF281(+),and PRRX1(+).Prog_Macro2 and Prog_Macro4 were located at the end of the developmental trajectory,and the prognostic ability of macrophage subsets increased with the progression of osteosarcoma.The prognostic model established based on the differentially expressed genes involved in macrophage differentiation can distinguish the survival rate of osteosarcoma patients with different risks(P<0.001).Conclusion Macrophage subsets are closely related to the prognosis of osteosarcoma and can be used as the key target cells for the immunotherapy of osteosarcoma.

MIF is a critical regulator of mononuclear phagocytic infiltration in hepatocellular carcinoma

Immunotherapy targeting tumor-associated macrophages (TAMs) is a promising approach to treating cancer. However, the limited drug targets and ambiguous mechanisms impede the development of clinical immunotherapy strategies. To elucidate the underlying processes involved in mononuclear phagocyte (MNP) infiltration and phenotypic changes in hepatocellular carcinoma (HCC), we integrated single-cell RNA-sequencing data from 100,030 cells derived from patients with HCC and healthy individuals and compared the phenotypes and origins of the MNPs in the tumor core, tumor periphery, adjacent normal tissue, and healthy liver samples. Using machine learning and multi-omics analyses, we identified 445 infiltration-associated genes and potential drug targets affecting this process. Through invitro experiments, we found that the expression of macrophage migration inhibitory factor (MIF) is the upstream regulator of secreted phosphoprotein 1 (SPP1) and promote migration in TAMs. Our findings also indicate that MIF promotes tumor metastasis and invasion and is a promising potential target for treating HCC.

Intrahepatic infiltration of activated CD8+ T cells and mononuclear phagocyte is associated with idiosyncratic drug-induced liver injury.

Idiosyncratic drug-induced liver injury (DILI) is caused by the interplay among drugs, their metabolites, and the host immune response. The characterization of infiltrated immune cells in the liver may improve the understanding of the pathogenesis of idiosyncratic DILI. This study investigated the phenotypes and clinical implications of liver-infiltrating immune cells in idiosyncratic DILI. From January 2017 to June 2021, 53 patients with idiosyncratic DILI who underwent liver biopsy were prospectively enrolled in this study. Immunohistochemical staining and flow cytometry analyses were performed on the biopsy specimens. Serum levels of CXC chemokine ligand 10 (CXCL10) and soluble CD163 were measured. A multivariate cox proportional hazards model was used to evaluate predictors of DILI resolution within 30 days. The numbers of intrahepatic T cells and mononuclear phagocytes were positively correlated with serum levels of total bilirubin, alanine aminotransferase (ALT), and the model of end-stage liver disease score. The frequency of activated CD8+ T cells among liver-infiltrating CD8+ T cells in DILI livers was higher than that in healthy livers. Notably, the percentages of activated intrahepatic CD8+ T cells and mononuclear phagocytes in DILI livers showed a positive correlation with ALT. Additionally, serum CXCL10 level was positively correlated with intrahepatic T cell infiltration and ALT, and soluble CD163 level was positively correlated with intrahepatic mononuclear phagocyte infiltration and ALT. Thirty-six patients (70.6%) were treated with steroids. In multivariate analysis, total bilirubin and steroid use independently influenced DILI resolution within 30 days. Activated CD8+ T cells and mononuclear phagocyte are associated with liver injury caused by drugs. Therefore, we suggest that steroids are a potential treatment option for idiosyncratic DILI.

Tenofovir alafenamide alleviates nonalcoholic steatohepatitis in mice by blocking the phosphorylation of AKT in intrahepatic mononuclear phagocytes

Background and purposeAlthough the prevalence of nonalcoholic steatohepatitis (NASH) is rapidly increasing, effective therapy is lacking. Tenofovir alafenamide (TAF) is a widely used antiviral drug for hepatitis B. In this study, we investigated the potential pharmacological effects of TAF on NASH. Experimental approachTwo different NASH mouse models were established: 1) by subcutaneous injection of streptozotocin (0.2 mg) and feeding the mice a high-fat, high-cholesterol (HFHC) diet, and 2) feeding the mice a choline-deficient, L-amino acid-defined, high-fat (CDAHF) diet. Key resultsSerum alanine aminotransferase and triglyceride levels in TAF-treated NASH mice were significantly lower than those in the mock-treated ones. The livers from the TAF-treated NASH mice showed attenuated mononuclear phagocyte (MP) infiltration compared to those from the mock-treated ones. TAF-treated NASH mice exhibited decreased liver infiltration of activated MPs (IAIE+/PD-L1+/MerTK+). In ex vivo experiments using sorted human CD14+ monocytes treated with lipopolysaccharide (LPS) and/or TAF, we confirmed the decreased level of phosphorylated AKT in TAF-treated LPS-stimulated monocytes compared to that in the mock-treated ones. Mouse liver immunoblotting showed that phosphorylation levels of AKT were significantly lower in the TAF-treated NASH group than in the mock-treated group. Conclusion and implicationsTAF exerts anti-inflammatory effects in NASH livers by attenuating AKT phosphorylation in intrahepatic activated MPs. Therefore, TAF may serve as a new therapeutic option for NASH.

Amount of Mononuclear Phagocyte Infiltrate Does Not Predict Area of Experimental Choroidal Neovascularization (CNV).

Purpose: Mononuclear phagocytes (MNPs) are present in neovascular age-related macular degeneration (nv AMD) which is also called choroidal neovascularization (CNV). The number and phenotype of the MNPs depend upon the local environment in the CNV and effect of nv AMD therapy. We investigated ocular cell infiltration and conditions that modulate angiogenesis in a laser-induced mouse CNV model.Methods: We developed assays to quantify MNPs in our established mouse CNV model. One MNP assay quantified the number of subretinal cells peripheral to the CNV lesions. A second assay semiquantitatively assesses the number of MNPs localized to the CNV lesion. We used these assays to measure the effect of toll-like receptor-2 (TLR-2) activation, anti-vascular endothelial growth factor (VEGF) therapy, and chemokine (C-C motif) ligand 2 (Ccl2) genetic deletion on MNP infiltration after laser injury.Results: Laser injury induced blood vessel growth and infiltration of MNPs. Systemic administration of a TLR-2 activating peptide increased laser-induced CNV area, MNP cell numbers, and MNP density over the CNV lesions. Systemic administration of a VEGF antibody reduced CNV area, while Ccl2 genetic deletion increased CNV area. Despite the change in amount of angiogenesis, MNP infiltration was, surprisingly, unchanged in these 2 conditions.Conclusions: MNP quantification provides biological insights for candidate AMD therapies. The number of infiltrating MNP cells does not correlate with the amount of laser-induced CNV area.

Aldosterone Exposure Causes Increased Retinal Edema and Severe Retinopathy Following Laser-Induced Retinal Vein Occlusion in Mice.

To determine the effects of aldosterone exposure on retinal edema and retinopathy in a mouse model of retinal vein occlusion (RVO). RVO was induced immediately following intravenous injection of Rose bengal (66 mg/kg) using a 532-nm wavelength laser to place three to seven applications at 80 mW and 50-μm spot size directed at the superior retinal vein one disc diameter away from the nerve. Negative control consisted of placing an equal number of laser spots without targeting the vein. Male and female C57BL/6J mice aged 7 to 9 months with confirmed absence of Crb1rd8 were used. Aldosterone pellets releasing a daily dose of 0.83 μg/day were implanted subcutaneously 4 weeks prior to RVO. Retinal imaging by optical coherence tomography (OCT) was performed using a Micron IV rodent imaging system. Retinas were analyzed by immunohistochemistry using standard techniques. Retinal imaging and tissue analysis were performed 2, 4, and 7 days following RVO. Comparisons were made using Student's t-test, ANOVA, and Pearson's χ2. RVO caused retinal edema in the form of cystic spaces and retinal thickening detectable by both OCT and histology. RVO also caused Müller glia (MG) dysfunction manifest as upregulated glial fibrillary acidic protein (GFAP) and altered localization of aquaporin 4 (AQP4) and Kir4.1. Treatment with aldosterone caused a significant increase in retinal edema and more severe retinopathy manifest as retinal whitening and extensive intraretinal hemorrhage. MG dysfunction was more severe and persistent in aldosterone-treated mice. Finally, aldosterone greatly increased the number of infiltrating mononuclear phagocytes following RVO. Systemic aldosterone exposure causes a more severe RVO phenotype manifest as increased severity and duration of retinal edema and more severe retinopathy. The effects of aldosterone may be mediated by MG dysfunction and increased infiltration of mononuclear phagocytes. This suggests that small increases in aldosterone levels may be a risk factor for severe RVO.

Inflammatory Resolution Triggers Mononuclear Phagocyte Infiltration, Which through COX-1/mPGES-1 Maintains Immune Tolerance

While resolution is believed to switch inflammation off and return the affected tissues back to homeostasis, we found that it triggers a prolonged phase of immune suppression. During resolution of acute inflammation (∼72h) CD4+ and CD8+ lymphocytes as well as natural killer (NK) cells migrated into tissues secreting IFNγ from day 6. IFNγ carried out two distinct functions: firstly, through IP-10, IFNγ indirectly triggered monocyte infiltration, which differentiated into macrophages (moMϕs). Secondly, moMϕs were directly acted upon by IFNγ to express microsomal prostaglandin E synthase-1 (mPGES-1) alongside cyclooxygenase (COX 1) resulting in sustained prostaglandin (PG)E2 synthesis peaking at day 14 and lasting for several weeks. PGE2 suppressed innate immune responses to bacterial infection opening a prolonged window of postresolution infectious opportunity within the host. At the same time, it also differentially modulated multiple aspect of the adaptive immune system including suppressing lymphocyte function whilst also generating myeloid-derived suppressor cells, the net effect of which was impaired uptake/presentation of exogenous antigens. Therefore, we have defined a novel sequence of post-resolution events starting with IFNγ and culminating in sustained PGE2, which we propose dampens the propensity to develop autoimmune responses to endogenous antigens at the cost of local tissue infection.

Macrophage-Derived Extracellular Succinate Licenses Neural Stem Cells to Suppress Chronic Neuroinflammation.

SummaryNeural stem cell (NSC) transplantation can influence immune responses and suppress inflammation in the CNS. Metabolites, such as succinate, modulate the phenotype and function of immune cells, but whether and how NSCs are also activated by such immunometabolites to control immunoreactivity and inflammatory responses is unclear. Here, we show that transplanted somatic and directly induced NSCs ameliorate chronic CNS inflammation by reducing succinate levels in the cerebrospinal fluid, thereby decreasing mononuclear phagocyte (MP) infiltration and secondary CNS damage. Inflammatory MPs release succinate, which activates succinate receptor 1 (SUCNR1)/GPR91 on NSCs, leading them to secrete prostaglandin E2 and scavenge extracellular succinate with consequential anti-inflammatory effects. Thus, our work reveals an unexpected role for the succinate-SUCNR1 axis in somatic and directly induced NSCs, which controls the response of stem cells to inflammatory metabolic signals released by type 1 MPs in the chronically inflamed brain.

Quantitative Analysis of Immune Cell Subset Infiltration of Supraspinatus Muscle After Severe Rotator Cuff Injury

Rotator cuff tears cause muscle degeneration that is characterized by myofiber atrophy, fatty infiltration, and fibrosis and is minimally responsive to current treatment options. The underlying pathogenesis of rotator cuff muscle degeneration remains to be elucidated, and increasing evidence implicates immune cell infiltration as a significant factor. Because immune cells are comprised of highly heterogeneous subpopulations that exert divergent effects on injured tissue, understanding trafficking and accumulation of immune subpopulations may hold the key to more effective therapies. The present study quantifies subpopulations of immune cells infiltrating the murine supraspinatus muscle after severe rotator cuff injury that includes tenotomy and denervation. Rotator cuff injury stimulates dramatic infiltration of mononuclear phagocytes, enriches mononuclear phagocytes in non-classical subpopulations, and enriches T lymphocytes in TH and Treg subpopulations. The combination of tenotomy plus denervation significantly increases mononuclear phagocyte infiltration, enriches macrophages in the non-classical subpopulation, and decreases T lymphocyte enrichment in TH cells compared to tenotomy alone. Depletion of circulating monocytes via liposomal clodronate accelerates supraspinatus atrophy after tenotomy and denervation. The study may aid rational design of immunologically smart therapies that harness immune cells to enhance outcomes after rotator cuff tears.

Subretinal mononuclear phagocytes induce cone segment loss via IL-1β.

Photo-transduction in cone segments (CS) is crucial for high acuity daytime vision. For ill-defined reasons, CS degenerate in retinitis pigmentosa (RP) and in the transitional zone (TZ) of atrophic zones (AZ), which characterize geographic atrophy (GA). Our experiments confirm the loss of cone segments (CS) in the TZ of patients with GA and show their association with subretinal CD14(+)mononuclear phagocyte (MP) infiltration that is also reported in RP. Using human and mouse MPs in vitro and inflammation-prone Cx3cr1(GFP/GFP) mice in vivo, we demonstrate that MP-derived IL-1β leads to severe CS degeneration. Our results strongly suggest that subretinal MP accumulation participates in the observed pathological photoreceptor changes in these diseases. Inhibiting subretinal MP accumulation or Il-1β might protect the CS and help preserve high acuity daytime vision in conditions characterized by subretinal inflammation, such as AMD and RP.

Lung Inflammation and Pulmonary Intravascular Macrophage Recruitment in L‐Arginine‐Induced Acute Necrotizing Pancreatitis in Mouse

Human patients suffering from severe acute pancreatitis are at higher risk of developing acute lung injury with high morbidity and mortality rates. Although there are many studies on the mechanisms leading to acute lung injury associated with acute pancreatitis in experimental models, there has not been any investigation on the induction of pulmonary intravascular macrophages (PIMs), a pro‐inflammatory cell normally present in cattle, pig, and horses, during this pathological condition in animal models. Therefore, we tested the hypothesis that lungs from mice with L‐arginine‐induced acute necrotizing pancreatitis (ANP) will be inflamed along with the recruitment of PIMs, compared to lungs of control mice. ANP was induced in C57BL/6 mice by administering two intraperitoneal injections, one hour apart, of a sterile L‐arginine monohydrochloride 9% solution at the dose of 4.5g/kg per injection, and mice were euthanized at 24h (n=7), 72h (n=7) and 120h (n=7) post‐injections. Control mice (n=9) received the same injections but with physiological sterile saline. ANP was confirmed by a pathologist using a histological grading system for pancreatic necrosis, infiltration of inflammatory cells and edema, which generated total ANP severity scores as follows: median (with range) of 0 for controls, 1 (0–3) for the 24h group, 8 (6–10) for the 72h group and 4 (2–10) for the 120h group. Serum amylase was significantly increased in the 24h group (5528±1210 U/L) and the 72h group (6547±1275 U/L), but not in the 120h group (1399±62 U/L), compared to control (1483±48 U/L), p&lt;0.001. The lung histological grading revealed infiltration of mononuclear phagocytes in the lungs of mice in the 72h group only, compared to the control lungs (p&lt;0.001). Immunohistochemistry with anti‐macrophage CD‐68 antibody revealed an increase of monocytes/macrophages present in alveolar septa of the lungs of all mice with ANP, suggesting PIMs induction, compared with control mice (p&lt;0.01), with cell counts (median; range) as follows: control mice (81; 55–147), 24h group (150; 115–190), 72h group (190; 118–200) and 120h group (135; 97–194). Bioplex assay on lung homogenates identified increased levels of IL‐6 (p &lt; 0,0001), IL‐10 (p &lt; 0,01), and MCP‐1 (p &lt; 0,05) in the 24h group only, compared to control mice. Immunohistochemistry for vWF revealed granular staining in the alveolar septal capillaries of some ANP mice, but not in control mice, suggesting activation of the lung microvasculature endothelium during ANP. The data show that lung inflammation in mice with L‐arginine‐induced ANP is initiated as early as 24h and is characterised by the infiltration of septal mononuclear phagocytes, most probably PIMs, as well as by increased lung concentrations of IL‐6, IL‐10 and MCP‐1. Because of the established role of PIMs in lung inflammation, the mouse model of ANP will be a useful tool to investigate the biology of PIMs in ANP‐associated lung inflammation.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada

Upregulation of P2RX7 in Cx3cr1-Deficient Mononuclear Phagocytes Leads to Increased Interleukin-1β Secretion and Photoreceptor Neurodegeneration.

Photoreceptor degeneration in age-related macular degeneration (AMD) is associated with an infiltration and chronic accumulation of mononuclear phagocytes (MPs). We have previously shown that Cx3cr1-deficient mice develop age- and stress- related subretinal accumulation of MPs, which is associated with photoreceptor degeneration. Cx3cr1-deficient MPs have been shown to increase neuronal apoptosis through IL-1β in neuroinflammation of the brain. The reason for increased IL-1β secretion from Cx3cr1-deficient MPs, and whether IL-1β is responsible for increased photoreceptor apoptosis in Cx3cr1-deficient mice, has not been elucidated. Here we show that Cx3cr1-deficient MPs express increased surface P2X7 receptor (P2RX7), which stimulates IL-1β maturation and secretion. P2RX7 and IL-1β inhibition efficiently blunted Cx3cr1-MP-dependent photoreceptor apoptosis in a monocyte/retina coculture system and in light-induced subretinal inflammation of Cx3cr1-deficient mice in vivo. Our results provide an explanation for increased CX3CR1-dependent IL-1β secretion and suggest that IL-1β or P2RX7 inhibition can help inhibit the inflammation-associated photoreceptor cell loss in late AMD, including geographic atrophy, for which no efficient treatment currently exists.

CC Chemokine Ligand 18 in ANCA-Associated Crescentic GN.

ANCA-associated vasculitis is the most frequent cause of crescentic GN. To define new molecular and/or cellular biomarkers of this disease in the kidney, we performed microarray analyses of renal biopsy samples from patients with ANCA-associated crescentic GN. Expression profiles were correlated with clinical data in a prospective study of patients with renal ANCA disease. CC chemokine ligand 18 (CCL18), acting through CC chemokine receptor 8 (CCR8) on mononuclear cells, was identified as the most upregulated chemotactic cytokine in patients with newly diagnosed ANCA-associated crescentic GN. Macrophages and myeloid dendritic cells in the kidney were detected as CCL18-producing cells. The density of CCL18(+) cells correlated with crescent formation, interstitial inflammation, and impairment of renal function. CCL18 protein levels were higher in sera of patients with renal ANCA disease compared with those in sera of patients with other forms of crescentic GN. CCL18 serum levels were higher in patients who suffered from ANCA-associated renal relapses compared with those in patients who remained in remission. Using a murine model of crescentic GN, we explored the effects of the CCL18 murine functional analog CCL8 and its receptor CCR8 on kidney function and morphology. Compared with wild-type mice, Ccr8(-/-) mice had significantly less infiltration of pathogenic mononuclear phagocytes. Furthermore, Ccr8(-/-) mice maintained renal function better and had reduced renal tissue injury. In summary, our data indicate that CCL18 drives renal inflammation through CCR8-expressing cells and could serve as a biomarker for disease activity and renal relapse in ANCA-associated crescentic GN.

Induction of macrophage migration by neurotoxic prion protein fragment

Prion diseases are characterized by accumulation of protease resistant isoforms of prion protein (PrP), and infiltration and activation of mononuclear phagocytes at the brain lesions. Interactions between prion proteins and immune cells during disease progression are still not very well understood. In the present study, multiwell chamber chemotaxis assay was carried out to assess the migratory response of macrophage cell line Ana-1 to a synthetic peptide homologous to residues 106–126 of the human prion protein. Specific protein kinase inhibitors were used to elucidate the signaling events underlying PrP106–126-induced macrophages migration, and a comparison with the signaling pattern of macrophage migration induced by substance P (SP) and N-formyl-methionyl-leucyl-phenylalanine (fMLP), respectively, was carried out. The results showed that PrP106–126 had a potent chemotactic effect on murine macrophage cell line Ana-1; that multiple signaling pathways might be involved in the PrP106–126-induced macrophage migrations; and that PrP106–126-induced chemotactic activity was similar to that induced by SP. These findings provide new insights into the mechanisms underlying the interaction between PrP and macrophages.

Liver injury caused by antibodies against dengue virus nonstructural protein 1 in a murine model

Clinical manifestations of severe dengue diseases include thrombocytopenia, vascular leakage, and liver damage. Evidence shows that hepatic injury is involved in the pathogenesis of dengue infection; however, the mechanisms are not fully resolved. Our previous in vitro studies suggested a mechanism of molecular mimicry in which antibodies directed against dengue virus (DV) nonstructural protein 1 (NS1) cross-reacted with endothelial cells and caused inflammatory activation and apoptosis. In this study, the pathogenic effects of anti-DV NS1 antibodies were further examined in a murine model. We found, in liver sections, that anti-DV NS1 antibodies bound to naive mouse vessel endothelium and the binding activity was inhibited by preabsorption of antibodies with DV NS1. Active immunization with DV NS1 resulted in antibody deposition to liver vessel endothelium, and also apoptotic cell death of liver endothelium. Liver tissue damage was observed in DV NS1-immunized mice by histological examination. The serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were increased in mice either actively immunized with DV NS1 protein or passively immunized with antibodies obtained from DV NS1-immunized mice. Furthermore, histological examination revealed mononuclear phagocyte infiltration and cell apoptosis in mice passively immunized with antibodies obtained from mice immunized with DV NS1. Increased AST and ALT levels were observed in mice passively immunized with purified immunoglobulin G (IgG) from dengue patients compared with normal control human IgG-immunized mice. The increased AST and ALT levels were inhibited when dengue patient serum IgG was preabsorbed with DV NS1. In conclusion, active immunization with DV NS1 protein causes immune-mediated liver injury in mice. Passive immunization provides additional evidence that anti-DV NS1 antibodies may play a role in liver damage, which is a pathologic manifestation in dengue virus disease.

Orchestration of the inflammatory response in ischemia‐reperfusion injury

Ischemia to nerve can cause fiber degeneration and reperfusion following ischemia [ischemia-reperfusion (IR)] adds the additional insult of an inflammatory response and oxidative injury. Limited information is available on the molecular mediators and their endoneurial targets. In this study, using a highly reproducible animal model of IR injury to nerve and selective immunolabeling methods [for nuclear factor kappa B (NF-kappaB), intercellular adhesion molecule-1 (ICAM-1), cytokines, and inflammatory cells] over an expanded time frame, we evaluated the temporal pattern and localization of mediators of the inflammatory response. Sixty rats were used. Nine groups (N=6 each) underwent complete hind limb ischemia for 4 h, followed by reperfusion durations of 0, 3, 12, 24, and 48 h, and 7, 14, 28, and 42 days. One group underwent sham operation (N=6). The earliest change was ICAM-1 expression in the microvessel (endothelial cell) followed almost immediately by NF-kappaB activation with axonal expression (24 and 48 h), followed by endoneurial edema and ischemic fiber degeneration (7 and 14 days). Granulocytic infiltration was followed by endoneurial infiltration of mononuclear phagocytes (14 days), expression of interleukin 6 (IL-6) (microvessels), and subsequent Schwann cell NF-kappaB expression. Granulocytes, tumor necrosis factor alpha, and IL-6-positive cells were observed primarily within the epineurium. IR results in changes in a number of interacting networks of targets and inflammatory mediators. NF-kappaB activation has a central orchestrating role involving both the axon and the Schwann cell in effecting the inflammatory response.

Multiple effects of hypothermia on inflammatory response following ischemia–reperfusion injury in experimental ischemic neuropathy

Hypothermia is neuroprotective in peripheral nerve ischemia, but the mechanism(s) of neuroprotection are not well known. A major mechanism of ischemia–reperfusion (IR) injury is the inflammatory response. We therefore dissected the effects of hypothermia on inflammatory mediators in peripheral nerve ischemia of rats. Following functional and pathological evaluations for the effect of hypothermia on IR injury, we undertook immunohistochemical studies of inflammatory cells, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), intercellular adhesion molecule-1 (ICAM-1), and nuclear factor kappa B (NF-κB) in nerve subjected to IR under defined hypothermic conditions with varying time delays (0, 1, 3, and 4 h) and depth of hypothermia (28°C, 32°C, and 35°C). Functionally and pathologically, significant hypothermic neuroprotection was confirmed in the intraischemically treated groups but not in the postischemically treated groups. In endoneurial microvessels, intraischemic hypothermia inhibited ICAM-1 upregulation but not TNF-α, NF-κB, and IL-6 expressions. We demonstrated significantly reduced granulocyte and mononuclear phagocyte infiltration into nerve with intraischemic hypothermia but not with postischemic hypothermia. Cytokine (TNF-α and IL-6) positive cells were significantly decreased in both epineurium and endoneurium with intraischemic hypothermia. Excess NF-κB expression was seen in both Schwann cell and axon under normothermia (35°C) but was inhibitable with deep hypothermia (28°C). We conclude that intraischemic hypothermia significantly attenuates the inflammatory response by its effect on multiple key mediators including cytokines, ICAM-1, and NF-κB.

Activated Monocytes Induce Human Retinal Pigment Epithelial Cell Apoptosis Through Caspase-3 Activation

Dysfunction and loss of human retinal pigment epithelial (HRPE) cells is a significant component of many ocular diseases, in which mononuclear phagocyte infiltration at the HRPE-related interface is also observed. In this study, we investigated whether HRPE cell apoptosis may be induced by overlay of IFN-γ–activated monocytes. Human monocytes primed with IFN-γ overlaid directly onto HRPE cells elicited significant increases in terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)–positive HRPE cells (p < 0.0001) and decreases of proliferating cell nuclear antigen–positive (p < 0.0001) HRPE cells. The activated monocytes also induced HRPE cell caspase-3 activation, which was inhibited by the caspase-3 inhibitor, Z-DEVD-fmk. However, co-incubations in which activated monocytes were prevented from direct contact with HRPE cells or in which the monocytes were separated from the HRPE cells after 30 minutes of direct contact, did not induce significant HRPE cell apoptosis. Function-blocking anti-CD18 and anti-intercellular adhesion molecule-1 (ICAM-1) antibodies significantly reduced activated monocyte-induced TUNEL-positive HRPE cells by 48% (p = 0.0051) and 38% (p = 0.046), respectively. Anti-CD18 and anti-ICAM-1 antibodies significantly inhibited caspase-3 activity by 56% (p < 0.0001) and 45% (p < 0.0001), respectively. However, antibodies to vascular cell adhesion molecule-1, TNF-α, IL-1β, or TNF-related apoptosis-inducing ligand did not inhibit apoptosis or caspase-3 activation. Direct overlay of monocytes also induced reactive oxygen metabolites (ROM) within HRPE cells. The intracellular HRPE cell ROM production was inhibited by the anti-CD18 and anti-ICAM-1 antibodies, but not by superoxide dismutase, presumably due to its failure to penetrate into HRPE cells. Accordingly, neither superoxide dismutase nor NG-monomethyl-l-arginine had significant effects on HRPE cell apoptosis or caspase-3 activation. Our results suggest that activated monocytes may induce ROM in HRPE cells through cell-to-cell contact, in part via CD18 and ICAM-1, and promote HRPE cell apoptosis. These mechanisms may compromise HRPE cell function and survival in a variety of retinal diseases.

The neurotoxic prion peptide fragment PrP(106-126) is a chemotactic agonist for the G protein-coupled receptor formyl peptide receptor-like 1.

Prion diseases are transmissible and fatal neurodegenerative disorders which involve infiltration and activation of mononuclear phagocytes at the brain lesions. A 20-aa acid fragment of the human cellular prion protein, PrP(106-126), was reported to mimic the biological activity of the pathologic isoform of prion and activates mononuclear phagocytes. The cell surface receptor(s) mediating the activity of PrP(106-126) is unknown. In this study, we show that PrP(106-126) is chemotactic for human monocytes through the use of a G protein-coupled receptor formyl peptide receptor-like 1 (FPRL1), which has been reported to interact with a diverse array of exogenous or endogenous ligands. Upon stimulation by PrP(106-126), FPRL1 underwent a rapid internalization and, furthermore, PrP(106-126) enhanced monocyte production of proinflammatory cytokines, which was inhibited by pertussis toxin. Thus, FPRL1 may act as a "pattern recognition" receptor that interacts with multiple pathologic agents and may be involved in the proinflammatory process of prion diseases.