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1.
Sci Rep ; 10(1): 13124, 2020 08 04.
Article in English | MEDLINE | ID: mdl-32753665

ABSTRACT

We recently showed that the anti-helminthic compound mebendazole (MBZ) has immunomodulating activity in monocyte/macrophage models and induces ERK signalling. In the present study we investigated whether MBZ induced ERK activation is shared by other tubulin binding agents (TBAs) and if it is observable also in other human cell types. Curated gene signatures for a panel of TBAs in the LINCS Connectivity Map (CMap) database showed a unique strong negative correlation of MBZ with MEK/ERK inhibitors indicating ERK activation also in non-haematological cell lines. L1000 gene expression signatures for MBZ treated THP-1 monocytes also connected negatively to MEK inhibitors. MEK/ERK phosphoprotein activity testing of a number of TBAs showed that only MBZ increased the activity in both THP-1 monocytes and PMA differentiated macrophages. Distal effects on ERK phosphorylation of the substrate P90RSK and release of IL1B followed the same pattern. The effect of MBZ on MEK/ERK phosphorylation was inhibited by RAF/MEK/ERK inhibitors in THP-1 models, CD3/IL2 stimulated PBMCs and a MAPK reporter HEK-293 cell line. MBZ was also shown to increase ERK activity in CD4+ T-cells from lupus patients with known defective ERK signalling. Given these mechanistic features MBZ is suggested suitable for treatment of diseases characterized by defective ERK signalling, notably difficult to treat autoimmune diseases.


Subject(s)
Extracellular Signal-Regulated MAP Kinases/metabolism , MAP Kinase Signaling System/drug effects , Mebendazole/pharmacology , Mitogen-Activated Protein Kinase Kinases/metabolism , Tubulin/metabolism , HEK293 Cells , Humans
2.
ACS Omega ; 5(28): 17242-17254, 2020 Jul 21.
Article in English | MEDLINE | ID: mdl-32715210

ABSTRACT

We have previously identified selective upregulation of the mevalonate pathway genes upon inhibition of oxidative phosphorylation (OXPHOS) in quiescent cancer cells. Using mass spectrometry-based proteomics, we here investigated whether these responses are corroborated on the protein level and whether proteomics could yield unique insights into context-dependent biology. HCT116 colon carcinoma cells were cultured as monolayer cultures, proliferative multicellular tumor spheroids (P-MCTS), or quiescent (Q-MCTS) multicellular tumor spheroids and exposed to OXPHOS inhibitors: nitazoxanide, FCCP, oligomycin, and salinomycin or the HMG-CoA-reductase inhibitor simvastatin at two different doses for 6 and 24 h. Samples were processed using an in-depth bottom-up proteomics workflow resulting in a total of 9286 identified protein groups. Gene set enrichment analysis showed profound differences between the three cell systems and confirmed differential enrichment of hypoxia, OXPHOS, and cell cycle progression-related protein responses in P-MCTS and Q-MCTS. Treatment experiments showed that the observed drug-induced alterations in gene expression of metabolically challenged cells are not translated directly to the protein level, but the results reaffirmed OXPHOS as a selective vulnerability of quiescent cancer cells. This work provides rationale for the use of deep proteome profiling to identify context-dependent treatment responses and encourages further studies investigating metabolic processes that could be co-targeted together with OXPHOS to eradicate quiescent cancer cells.

3.
BMC Res Notes ; 12(1): 234, 2019 Apr 22.
Article in English | MEDLINE | ID: mdl-31010428

ABSTRACT

OBJECTIVE: We recently showed that the anti-helminthic compound mebendazole (MBZ) has immunomodulating activity by inducing a M2 to M1 phenotype switch in monocyte/macrophage models. In the present study we investigated the potential role of protein kinases in mediating this effect. RESULTS: MBZ potently binds and inhibits Dual specificity tyrosine-phosphorylation-regulated kinase 1B (DYRK1B) with a Kd and an IC50 of 7 and 360 nM, respectively. The specific DYRK1B inhibitor AZ191 did not mimic the cytokine release profile of MBZ in untreated THP-1 monocytes. However, in THP-1 cells differentiated into macrophages, AZ191 strongly induced a pro-inflammatory cytokine release pattern similar to MBZ and LPS/IFNγ. Furthermore, like MBZ, AZ191 increased the expression of the M1 marker CD80 and decreased the M2 marker CD163 in THP-1 macrophages. In this model, AZ191 also increased phospho-ERK activity although to a lesser extent compared to MBZ. Taken together, the results demonstrate that DYRK1B inhibition could, at least partly, recapitulate immune responses induced by MBZ. Hence, DYRK1B inhibition induced by MBZ may be part of the mechanism of action to switch M2 to M1 macrophages.


Subject(s)
Antinematodal Agents/pharmacology , Macrophages/drug effects , Mebendazole/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Antigens, CD/genetics , Antigens, CD/immunology , Antigens, Differentiation, Myelomonocytic/genetics , Antigens, Differentiation, Myelomonocytic/immunology , Antinematodal Agents/metabolism , Cell Differentiation/drug effects , Gene Expression Regulation , Heterocyclic Compounds, 2-Ring/pharmacology , Humans , Interferon-gamma/genetics , Interferon-gamma/immunology , Interferon-gamma/pharmacology , Interleukins/genetics , Interleukins/immunology , Lipopolysaccharides/pharmacology , Macrophages/cytology , Macrophages/immunology , Mebendazole/metabolism , Phosphorylation/drug effects , Protein Binding , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/immunology , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/immunology , Proto-Oncogene Proteins c-abl/genetics , Proto-Oncogene Proteins c-abl/immunology , Pyrimidines/pharmacology , Receptors, Cell Surface/genetics , Receptors, Cell Surface/immunology , Signal Transduction , THP-1 Cells , Tetradecanoylphorbol Acetate/pharmacology , Dyrk Kinases
4.
Oncotarget ; 9(56): 30805-30813, 2018 Jul 20.
Article in English | MEDLINE | ID: mdl-30112108

ABSTRACT

Mebendazole (MBZ) was recently shown to induce a tumor suppressive M1 phenotype in THP-1 monocytes and macrophages. In the present study the immune effects of MBZ was further investigated using human peripheral blood mononuclear cells (PBMCs) co-cultured with tumour cells. The Biomap platform was used to screen for biomarkers induced from MBZ exposed co-cultures of T-cell receptor activated PBMCs, HT29 colon cancer cells and either human fibroblasts or human umbilical vein endothelial cells (HUVEC) cells. In these co-culture systems MBZ at 0.3-10 µM induced significant increases in TNFα and IFNγ indicating immune stimulation. PBMC cultures alone were subsequently tested for activation status and only in PBMCs activated by CD3/IL2 stimulation and MBZ, at a clinically achievable concentration, was able to increase PBMC clustering and release of pro-inflammatory IFNγ, TNFα, IL6 and IL1ß cytokines. Moreover, when PBMC cultures were functionally tested for immune cell killing of lung cancer A549NucLightRed cells, MBZ significantly increased tumour cell apoptosis and reduced the number of surviving tumour cells. This effect was dependent on the presence of CD14 monocytes/macrophages in the co-culture. In summary, MBZ potentiated the immune stimulatory and anticancer effects of anti-CD3/IL2 activated PBMCs which could be relevant to explain the anticancer activity of MBZ observed in the clinic.

5.
Immunopharmacol Immunotoxicol ; 39(4): 199-210, 2017 Aug.
Article in English | MEDLINE | ID: mdl-28472897

ABSTRACT

Mebendazole (MBZ), a drug commonly used for helminitic infections, has recently gained substantial attention as a repositioning candidate for cancer treatment. However, the mechanism of action behind its anticancer activity remains unclear. To address this problem, we took advantage of the curated MBZ-induced gene expression signatures in the LINCS Connectivity Map (CMap) database. The analysis revealed strong negative correlation with MEK/ERK1/2 inhibitors. Moreover, several of the most upregulated genes in response to MBZ exposure were related to monocyte/macrophage activation. The MBZ-induced gene expression signature in the promyeloblastic HL-60 cell line was strongly enriched in genes involved in monocyte/macrophage pro-inflammatory (M1) activation. This was subsequently validated using MBZ-treated THP-1 monocytoid cells that demonstrated gene expression, surface markers and cytokine release characteristic of the M1 phenotype. At high concentrations MBZ substantially induced the release of IL-1ß and this was further potentiated by lipopolysaccharide (LPS). At low MBZ concentrations, cotreatment with LPS was required for MBZ-stimulated IL-1ß secretion to occur. Furthermore, we show that the activation of protein kinase C, ERK1/2 and NF-kappaB were required for MBZ-induced IL-1ß release. MBZ-induced IL-1ß release was found to be dependent on NLRP3 inflammasome activation and to involve TLR8 stimulation. Finally, MBZ induced tumor-suppressive effects in a coculture model with differentiated THP-1 macrophages and HT29 colon cancer cells. In summary, we report that MBZ induced a pro-inflammatory (M1) phenotype of monocytoid cells, which may, at least partly, explain MBZ's anticancer activity observed in animal tumor models and in the clinic.


Subject(s)
Antineoplastic Agents/pharmacology , Inflammasomes/drug effects , MAP Kinase Signaling System/drug effects , Macrophage Activation/drug effects , Mebendazole/pharmacology , Monocytes/drug effects , Toll-Like Receptor 8/metabolism , Cell Line , Cell Line, Tumor , Gene Expression/drug effects , HEK293 Cells , HL-60 Cells , HT29 Cells , Humans , Inflammasomes/metabolism , Interleukin-1beta/metabolism , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Macrophages/metabolism , Monocytes/metabolism , NF-kappa B/metabolism , Up-Regulation/drug effects
6.
Cell Chem Biol ; 23(11): 1428-1438, 2016 Nov 17.
Article in English | MEDLINE | ID: mdl-27984028

ABSTRACT

Cancer cell lines grown as two-dimensional (2D) cultures have been an essential model for studying cancer biology and anticancer drug discovery. However, 2D cancer cell cultures have major limitations, as they do not closely mimic the heterogeneity and tissue context of in vivo tumors. Developing three-dimensional (3D) cell cultures, such as multicellular tumor spheroids, has the potential to address some of these limitations. Here, we combined a high-throughput gene expression profiling method with a tumor spheroid-based drug-screening assay to identify context-dependent treatment responses. As a proof of concept, we examined drug responses of quiescent cancer cells to oxidative phosphorylation (OXPHOS) inhibitors. Use of multicellular tumor spheroids led to discovery that the mevalonate pathway is upregulated in quiescent cells during OXPHOS inhibition, and that OXPHOS inhibitors and mevalonate pathway inhibitors were synergistically toxic to quiescent spheroids. This work illustrates how 3D cellular models yield functional and mechanistic insights not accessible via 2D cultures.


Subject(s)
Antineoplastic Agents/pharmacology , Cell Culture Techniques/methods , Drug Screening Assays, Antitumor/methods , Neoplasms/drug therapy , Oxidative Phosphorylation/drug effects , Spheroids, Cellular/drug effects , Cell Line, Tumor , Gene Expression Profiling/methods , High-Throughput Screening Assays/methods , Humans , Mevalonic Acid/metabolism , Neoplasms/genetics , Neoplasms/metabolism , Signal Transduction/drug effects , Spheroids, Cellular/metabolism , Transcriptome/drug effects , Tumor Cells, Cultured
7.
FEBS J ; 281(24): 5432-46, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25271100

ABSTRACT

Knowledge on the contribution of protein glycosylation in host defense antimicrobial peptides is still scarce. We have studied here how the post-translational modification pattern modulates the antimicrobial activity of one of the best characterized leukocyte granule proteins. The human eosinophil cationic protein (ECP), an eosinophil specific granule protein secreted during inflammation and infection, can target a wide variety of pathogens. Previous work in human eosinophil extracts identified several ECP native forms and glycosylation heterogeneity was found to contribute to the protein biological properties. In this study we analyze for the first time the antimicrobial activity of the distinct native proteins purified from healthy donor blood. Low and heavy molecular weight forms were tested on Escherichia coli cell cultures and compared with the recombinant non-glycosylated protein. Further analysis on model membranes provided an insight towards an understanding of the protein behavior at the cytoplasmic membrane level. The results highlight the significant reduction in protein toxicity and bacteria agglutination activity for heavy glycosylated fractions. Notwithstanding, the lower glycosylated fraction mostly retains the lipopolysaccharide binding affinity together with the cytoplasmic membrane depolarization and membrane leakage activities. From structural analysis we propose that heavy glycosylation interferes with the protein self-aggregation, hindering the cell agglutination and membrane disruption processes. The results suggest the contribution of post-translational modifications to the antimicrobial role of ECP in host defense.


Subject(s)
Eosinophil Cationic Protein/physiology , Protein Processing, Post-Translational , Eosinophil Cationic Protein/metabolism , Eosinophil Cationic Protein/pharmacology , Escherichia coli/drug effects , Glycosylation , Humans , Microbial Sensitivity Tests
8.
Mol Immunol ; 55(3-4): 372-80, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23597768

ABSTRACT

Eosinophil cationic protein (ECP) is a toxic, granule-stored protein of the eosinophil granulocyte. It is a heterogeneous protein; molecular weights can differ from 15 to 22 kDa, due to glycosylations. We purified high molecular weight ECP from blood donors with the ECP434GG (rs2073342) genotype, with the aim of examining whether removal of carbohydrates could enhance the cytotoxic capacity. The cytotoxic activity of the ECP pools was tested against the NCI-H69 cell line, before and after enzymatic deglycosylation. ECP was also analysed by SELDI-TOF MS to monitor the changes in molecular mass after deglycosylation. Five high molecular weight pools of ECP (HMW-ECP I-V) with decreasing degrees of glycosylation were tested at concentrations ranging from 0.02 to 0.6 µM. The activity ranged from EC50 of >0.6 µM to 0.04 µM; HMW-ECP II had the lowest activity and HMW-ECP V the highest. After deglycosylation with N-glycosidase F, pools HMW-ECP I-III were reduced to the same molecular weight of 15.78 kDa and acquired potent cytotoxic activities. HMW-ECP IV and V with molecular species at 16.3 and 16.1 kDa were highly cytotoxic as such and were only partially deglycosylated, with slight enhancement of the toxic properties. The results suggest the presence of several HMW-ECP molecular species with differences in their post-translational modifications and cytotoxic properties. We conclude that a fraction of native ECP is stored in a non-cytotoxic form, which can be converted into a cytotoxic form by N-deglycosylation, whereas another fraction is stored as a highly cytotoxic form carrying different post-translational modifications.


Subject(s)
Asparagine/physiology , Cytotoxicity, Immunologic , Eosinophil Cationic Protein/toxicity , Polysaccharides/physiology , Protein Processing, Post-Translational/immunology , Cell Line, Tumor , Cytotoxicity, Immunologic/genetics , Eosinophil Cationic Protein/genetics , Genetic Variation , Glycosylation , Humans , Molecular Weight , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
9.
World J Gastroenterol ; 18(44): 6409-19; discussion p. 6417-8, 2012 Nov 28.
Article in English | MEDLINE | ID: mdl-23197886

ABSTRACT

AIM: To study the association between inflammatory bowel disease (IBD) and genetic variations in eosinophil protein X (EPX) and eosinophil cationic protein (ECP). METHODS: DNA was extracted from ethylene diamine tetraacetic acid blood of 587 patients with Crohn's disease (CD), 592 with ulcerative colitis (UC) and 300 healthy subjects. The EPX405 (G > C, rs2013109), ECP434 (G > C, rs2073342) and ECP562 (G > C, rs2233860) gene polymorphisms were analysed, by the 5'-nuclease allelic discrimination assay. For determination of intracellular content of EPX and ECP in granulocytes, 39 blood samples was collected and extracted with a buffer containing cetyltrimethylammonium bromide. The intracellular content of EPX was analysed using an enzyme-linked immunosorbent assay. The intracellular content of ECP was analysed with the UniCAP(®) system as described by the manufacturer. Statistical tests for calculations of results were χ(2) test, Fisher's exact test, ANOVA, Student-Newman-Keuls test, and Kaplan-Meier survival curve with Log-rank test for trend, the probability values of P < 0.05 were considered statistically significant. RESULTS: The genotype frequency for males with UC and with an age of disease onset of ≥ 45 years (n = 57) was for ECP434 and ECP562, GG = 37%, GC = 60%, CC = 4% and GG = 51%, GC = 49%, CC = 0% respectively. This was significantly different from the healthy subject's genotype frequencies of ECP434 (GG = 57%, GC = 38%, CC = 5%; P = 0.010) and ECP562 (GG = 68%, GC = 29%,CC = 3%; P = 0.009). The genotype frequencies for females, with an age of disease onset of ≥ 45 years with CD (n = 62), was for the ECP434 and ECP562 genotypes GG = 37%, GC = 52%, CC = 11% and GG = 48%, GC = 47% and CC = 5% respectively. This was also statistically different from healthy controls for both ECP434 (P = 0.010) and ECP562 (P = 0.013). The intracellular protein concentration of EPX and ECP was calculated in µg/10(6) eosinophils and then correlated to the EPX 405 genotypes. The protein content of EPX was highest in the patients with the CC genotype of EPX405 (GG = 4.65, GC = 5.93, and CC = 6.57) and for ECP in the patients with the GG genotype of EPX405 (GG = 2.70, GC = 2.47 and CC = 1.90). ANOVA test demonstrated a difference in intracellular protein content for EPX (P = 0.009) and ECP (P = 0.022). The age of disease onset was linked to haplotypes of the EPX405, ECP434 and ECP562 genotypes. Kaplan Maier curve showed a difference between haplotype distributions for the females with CD (P = 0.003). The highest age of disease onset was seen in females with the EPX405CC, ECP434GC, ECP562CC haplotype (34 years) and the lowest in females with the EPX405GC, ECP434GC, ECP562GG haplotype (21 years). For males with UC there was also a difference between the highest and lowest age of the disease onset (EPX405CC, ECP434CC, ECP562CC, mean 24 years vs EPX405GC, ECP434GC, ECP562GG, mean 34 years, P = 0.0009). The relative risk for UC patients with ECP434 or ECP562-GC/CC genotypes to develop dysplasia/cancer was 2.5 (95%CI: 1.2-5.4, P = 0.01) and 2.5 (95%CI: 1.1-5.4, P = 0.02) respectively, compared to patients carrying the GG-genotypes. CONCLUSION: Polymorphisms of EPX and ECP are associated to IBD in an age and gender dependent manner, suggesting an essential role of eosinophils in the pathophysiology of IBD.


Subject(s)
Colitis, Ulcerative/genetics , Crohn Disease/genetics , Eosinophil Cationic Protein/genetics , Eosinophil-Derived Neurotoxin/genetics , Eosinophils/enzymology , Polymorphism, Genetic , Adolescent , Adult , Age of Onset , Aged , Aged, 80 and over , Analysis of Variance , Case-Control Studies , Chi-Square Distribution , Colitis, Ulcerative/blood , Colitis, Ulcerative/enzymology , Colitis, Ulcerative/immunology , Crohn Disease/blood , Crohn Disease/enzymology , Crohn Disease/immunology , Eosinophil Cationic Protein/blood , Eosinophil-Derived Neurotoxin/blood , Eosinophils/immunology , Female , Gene Frequency , Genetic Predisposition to Disease , Haplotypes , Humans , Kaplan-Meier Estimate , Male , Middle Aged , Phenotype , Sex Factors , Sweden , Young Adult
10.
Eur J Haematol ; 87(3): 208-16, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21623917

ABSTRACT

OBJECTIVE: Classical Hodgkin's lymphoma (HL) lesions comprise few tumour cells, surrounded by numerous inflammatory cells. Like in other malignancies, the microenvironment is presumed to be clinically important in HL; however, microenvironment predictors remain poorly characterised. The aim of this study was to investigate how selected patient characteristics and genetic factors affect HL phenotype, in particular tissue eosinophilia, mast cell counts and HL histological subtype. METHODS: In a population-based study, patients with HL were interviewed about potential HL risk factors. Available tumours, n=448, were classified histologically; the number of eosinophils and mast cells were estimated, and eosinophil cationic protein (ECP) and eosinophil protein-x (EPX) gene polymorphisms were determined. Associations were assessed in regression models. RESULTS: Self-reported history of asthma was predictive of having tumour eosinophilia [≥200 eosinophils/10 high power fields, univariate odds ratio (OR)=2.22, 95% CI 1.06-4.64, P=0.03]. High numbers of eosinophils were predominantly seen in patients carrying the genotype ECP434GG [multivariate relative levels (RLs)=1.84, 95% CI 1.02-3.30, P=0.04]. Lower number of eosinophils was seen in Epstein-Barr virus (EBV)-positive tumours (univariate RL=0.52, 95% CI 0.3-0.9, P=0.02) and in older patients (univariate RL=0.85, 95% CI 0.73-0.99, P=0.03). Well-known factors such as young age, female sex and EBV-negative status predicted nodular sclerosis histology. CONCLUSION: The number of eosinophils in HL tumours is influenced by patient traits such as asthma, ECP genotype and EBV status. EBV status was predictive of histology.


Subject(s)
Eosinophils/pathology , Hodgkin Disease/epidemiology , Hodgkin Disease/pathology , Mast Cells/pathology , Asthma , Cell Count , Data Collection , Female , Genotype , Herpesvirus 4, Human , Humans , Male , Predictive Value of Tests , Prognosis , Risk Factors
11.
Exp Hematol ; 39(8): 850-8, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21679745

ABSTRACT

OBJECTIVE: In Hodgkin lymphoma (HL), tumor eosinophilia indicates poor prognosis, probably caused by eosinophil-induced stimulation of tumor cells. Our aim was to investigate the effects of eosinophil cationic protein (ECP) on HL tumor cells in vitro. MATERIALS AND METHODS: A fluorometric microculture cytotoxicity assay was used to measure the survival index of cells from the HL cell lines: HDLM-2 (T-cell origin, nodular sclerosis histology), KMH2 (B-cell origin, mixed cellularity), and L428 (B-cell origin, nodular sclerosis) after incubation with ECP97arg variants with different glycosylations and with ECP97thr. Flow cytometry monitored the effects of ECP on markers of cell death. RESULTS: For KMH2 and L428, ECP was cytotoxic with a dose-response relationship similar to a previously investigated small-cell lung cancer cell line. HDLM-2 was more sensitive to ECP at low concentrations, but reached a plateau (survival index of 70%) at 0.018 µM. The IC(50) for KMH2 and L428 were 0.2 and 0.15 µM, respectively. The IC(50) was never reached for HDLM-2. All tested ECP variants displayed similar activity in HDLM-2, in contrast to KMH2 and L428, which were more sensitive to less glycosylated ECP. Positive DNA staining (propidium iodide) of HDLM-2 cells treated with ECP indicated cell death by necrosis. CONCLUSIONS: ECP is cytotoxic for HL tumor cells even at low concentrations, but heterogeneity between cell lines exists and not all tumor cells are eradicated. Two cell lines of B-cell origin, KMH2 and L428, were sensitive to ECP in a dose-response manner, but for HDLM-2, which is of T-cell origin, the cytotoxicity reached a plateau.


Subject(s)
Eosinophil Cationic Protein/isolation & purification , Eosinophil Cationic Protein/pharmacology , Eosinophils/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Dose-Response Relationship, Drug , Eosinophil Cationic Protein/chemistry , Flow Cytometry , Glycosylation , Hodgkin Disease/metabolism , Hodgkin Disease/pathology , Humans , Inhibitory Concentration 50 , Ki-1 Antigen/metabolism , Molecular Weight , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
12.
Clin J Am Soc Nephrol ; 5(12): 2229-35, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20829422

ABSTRACT

BACKGROUND AND OBJECTIVES: Several molecular forms of human neutrophil lipocalin/neutrophil gelatinase-associated lipocalin (HNL/NGAL), a novel biomarker for acute kidney injury (AKI), have been found in urine. The origin of these different forms and the effect of antibody configuration on assay performances were investigated in this report. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: The molecular forms of HNL/NGAL from human neutrophils and present in urine obtained from cardiac surgery patients and patients with urinary tract infection (UTI), as well as secreted from HK-2 cells, were studied by Western blotting. The levels of HNL/NGAL in urine were measured by ELISAs. Kidney injury was simulated by incubation of HK-2 cells under stressful conditions. RESULTS: The major molecular form of HNL/NGAL secreted by neutrophils is dimeric, whereas the major form secreted by HK-2 cells is monomeric. This was reflected by a predominance of the monomeric form in urine from patients with AKI and the dimeric form in patients with UTIs. The epitope specificities of the antibody used in the ELISAs had a profound effect on assay performance and paralleled differences of the antibodies to identify the different forms of urine HNL/NGAL. CONCLUSIONS: The monomeric form is the predominant form secreted by tubular epithelial cells, and the dimeric form is the predominant form secreted by neutrophils. The development of molecular form-specific assays for HNL/NGAL may be a means to identify the origin of HNL/NGAL in urine and construct more specific tools for the diagnosis of AKI.


Subject(s)
Acute-Phase Proteins/urine , Lipocalins/urine , Proto-Oncogene Proteins/urine , Acute Kidney Injury/diagnosis , Acute Kidney Injury/urine , Acute-Phase Proteins/chemistry , Blotting, Western , Chromatography, Gel , Enzyme-Linked Immunosorbent Assay , Epithelial Cells/metabolism , Humans , Kidney/metabolism , Lipocalin-2 , Lipocalins/chemistry , Proto-Oncogene Proteins/chemistry
13.
J Immunol ; 183(6): 3949-54, 2009 Sep 15.
Article in English | MEDLINE | ID: mdl-19692640

ABSTRACT

The eosinophil granulocyte is an inflammatory cell involved in allergic diseases such as asthma and rhinitis. Eosinophil cationic protein (ECP) is a basic and potentially cytotoxic granule protein that is released from the eosinophil upon activation. The aim was to study secretion of molecular variants of ECP from blood eosinophils with the hypothesis that the stored noncytotoxic ECP is altered into cytotoxic species upon release from the cell. Eosinophil granulocytes were purified to >95% from venous blood from birch pollen allergic subjects, with symptoms of rhinitis, and from healthy control subjects during the birch pollen season. The cells were stimulated with IL-5, GM-CSF, or serum-opsonized Sephadex particles. Concentration of ECP in cells or supernatants was measured by means of a fluoroenzyme immunoassay, and ECP heterogeneity was studied using an affinity capture assay with the surface-enhanced laser desorption/ionization-time of flight mass spectrometry technique. Extracts of unstimulated eosinophils contained 10 major ECP variants, with molecular masses ranging from 16.1 to 17.7 kDa. Stimulation with particles mainly induced the secretion of two molecular variants at 16.1 and 16.3 kDa, while cytokine stimulation gave rise to a different secretion profile. ECP variants in the pellet extracts remained unaffected by cell activation. The modifications of secreted ECP were partly explained by differences in N-linked glycosylations. Secretion of ECP from eosinophils involves protein modification. The molecular masses of released ECP have acquired the masses of the cytotoxic species.


Subject(s)
Eosinophil Cationic Protein/metabolism , Eosinophils/metabolism , Betula , Case-Control Studies , Eosinophil Cationic Protein/analysis , Eosinophils/immunology , Glycosylation , Humans , Mass Spectrometry , Pollen , Protein Processing, Post-Translational , Rhinitis, Allergic, Seasonal/immunology
14.
J Immunol ; 183(1): 445-51, 2009 Jul 01.
Article in English | MEDLINE | ID: mdl-19542456

ABSTRACT

Eosinophil cationic protein (ECP) is a secretory protein of the eosinophil granulocyte, a cell involved in innate immunity. Functional studies have implicated ECP in numerous processes, such as tissue remodeling in allergic inflammation and cytotoxicity toward a variety of pathogens. Recent genetic studies have suggested that the ECP 434(G>C) polymorphism resulting in an arg97thr substitution would alter the function of ECP in vivo. Functional (in vitro) studies of ECP up until now have either been conducted with native preparations containing an unknown mixture of the ECP(97arg) and ECP(97thr) variants, or with recombinant proteins. Therefore, we have now for the first time extracted the native ECP(97arg) and ECP(97thr) variants from healthy blood donors and tested them functionally in vitro. Our results show that the arg97thr shift dramatically alters the cytotoxic capacity of ECP in vitro; the tested ECP(97arg) variants were cytotoxic toward the small-cell lung cancer cell line NCI-H69, whereas ECP(97thr) was noncytotoxic. RNase activity was unaffected by the arg97thr substitution. Both ECP(97arg) and ECP(97thr) stimulated fibroblast-mediated collagen gel contraction, an experimental model, which depicts wound healing, in a dose-dependent manner. In conclusion, our results demonstrate that the ECP 434(G>C) gene polymorphism affects the functional properties of native ECP, but also that there is a dissociation between different biological activities; the arg97thr substitution impairs the cytotoxic potential of ECP but less the gel contraction and not at all the RNase activity.


Subject(s)
Cytotoxicity, Immunologic/genetics , Eosinophil Cationic Protein/genetics , Eosinophil Cationic Protein/toxicity , Fibroblasts/physiology , Polymorphism, Genetic , Ribonucleases/metabolism , Amino Acid Substitution/genetics , Animals , Arginine/genetics , Cell Culture Techniques , Cell Line, Tumor , Cells, Cultured , Collagen Type I/metabolism , Eosinophil Cationic Protein/physiology , Gels , Genotype , Humans , Rats , Threonine/genetics
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