scholarly journals Glucocorticoid Regulation of Erythropoiesis in Humans: A Study of Patients with Cushing's Disease

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2135-2135
Author(s):  
Eliza B Geer ◽  
Lilian Varricchio ◽  
Fabrizio Martelli ◽  
Wu He ◽  
Lizette Couto ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cushing’S Disease ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Surface Expression ◽  
Cushing's Disease ◽  
Cell Surface Expression ◽  
Cd34 Cells ◽  
Mean Time ◽  
Over Time

Abstract Cushing's disease (CD) is a rare endocrine disorder (1.2-2.4/million/year) characterized by chronic excess endogenous glucocorticoids (GC) due to an adrenocorticotropic hormone-secreting pituitary adenoma. Untreated CD results in increased mortality and multiple morbidities (obesity, diabetes, hypertension, cardiovascular disease) and, in one case report, erythrocytosis (Gursoy et al, J End Invest. 2006;29:742). The effects of chronic GC exposure on erythropoiesis in a CD cohort have not yet been studied. We prospectively quantified hematocrit (Hct), hemoglobin (Hb) and platelets (ptl) values in CD patients before (v1) and after surgical remission (v2, mean time since surgery=14.5 months) and in matched healthy controls (HC). Frequency, antigenic profiling and erythroid (Ery) expansion potential of circulating hematopoietic progenitor cells (HPC) in the three cohorts were also evaluated. The subjects analyzed included 28 v1 [6 males, 22 females, mean age=41 years; body mass index (BMI)=33.3 kg/m2 ] and 13 HC [2 males, 11 females, mean age=41 years; BMI=30.7 kg/m2 (range 26.7-34.7, p=0.073). Eleven patients were analyzed over time in both v1 and v2. Mean cortisol in v1 (22.2±6.3 ug/dL) was higher than in HC (8.5±3.8 ug/dL, p=3.4E-07) and decreased in v2 (17.6±5.1% vs. 8.7±3.9%, N= 11, p=0.0006). Hct was higher in v1 than in HC (39.8±4.7%, vs. 38.8±2.7%, p = 0.045). In the 11 patients analyzed over time, hct decreased in v2 vs. v1 (39.2±4.7% vs. 42.3±4.4%, p=0.011). Hb in v1 was not different than in HC (13.10±1.6 vs. 13.12±3.8 g/dL, p =0.225) but decreased in patients studied both in v1 and V2 (14.1±1.5 g/dL vs. 13.2±1.8 g/dL, p=0.009). Similarly, plts were not different in v1 and HC (272.3±81.4 vs. 240.8±76.0 K/uL, p=0.274) but decreased in v2 (307.7±112.1 vs. 270.6±74.5 K/uL, p=0.021). In v1, Hct did not correlate with serum (R = 0.34, p = 0.33) or 24h urine (R = 0.072, p = 0.73) cortisol concentrations. There was no difference in frequency of HPC among v1, v2 and HC [2.6±3.0, 0.34±0.28 and 1.26±0.67% of CD34+ cells and 30.2±27.2, 23.7±13.2 and 16.5±11.5 CFC/105 mononuclear cells (MNC) in v1, v2 and HC]. CD34pos cells from all groups expressed similar levels of cKIT, IL-3Rβ and prominin1, but a greater proportion of those from v1 and v2 expressed thrombospondin and thrombopoietin (Mpl) receptors than those from HC (2 vs 0.4%), suggesting that CD HPC are biased toward erythro-megakaryocytopoiesis. Consistently, in cultures without the synthetic GC dexamethasone (Dex), MNC from v1 (12±6 %) and v2 (19%) generated in 10 days a greater proportion of Erys than MNC from HC (2±1% ). However, in cultures with Dex, MNC from v1 (48±25), but not those from v2 (70±23), generated less Erys than MNC from HC (83±26 , p=0.03), suggesting that Erys from v1 HPC respond poorly to Dex. This was tested by comparing the ability of Dex to induce biochemical (GRα phosphorylation at S211 and cell-surface expression of CXCR4/calreticulin) and biological (proliferation in synergy with growth factors, GFs) responses in Erys from CD and HC. Erys from v1, v2 and HC expressed equivalent levels of GRα but those from v1 Erys contained lower levels of pGRαS211/S203 than those from v2 or HC (Fig 1A). In contrast with HC Erys, Dex decreased GRα and did not induce pGRαS211 in v1 Erys (Fig 1B). Moreover, Dex increased cell-surface expression of CXCR4 (MFI from 580 to 700) and calreticulin (MFI from 300 to 700) and proliferation (by 30%) in HC Erys but not in those from v1 s (CXCR4 MFI from 278 to 154, calreticulin MFI from 136 to 152; proliferation increases by 6%). These results indicate that chronic GC excess increases Hct values but may also activate a post-transcriptional mechanism that reduces GRα expression inducing desensitization of erythroid cells to GC. Figure 1. A) Levels of total, pS211 and pS203 GRα in Erys from HC, v1 and v2. B) levels of GRα and GRα phosphorylated at pS203 in Erys from HC and v1 exposed from 15' to Dex alone or in combination with the GR inhibitor RU486. Figure 1. A) Levels of total, pS211 and pS203 GRα in Erys from HC, v1 and v2. B) levels of GRα and GRα phosphorylated at pS203 in Erys from HC and v1 exposed from 15' to Dex alone or in combination with the GR inhibitor RU486. Disclosures No relevant conflicts of interest to declare.

Cell surface expression of LDL receptor in chronic hepatitis C: correlation with viral load

2007 ◽  
Vol 293 (1) ◽  
pp. E416-E420 ◽  
Author(s):  
Jean-Michel Petit ◽  
Anne Minello ◽  
Laurence Duvillard ◽  
Valérie Jooste ◽  
Serge Monier ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Hepatitis ◽  
Viral Load ◽  
Hepatitis C ◽  
Cell Surface ◽  
Chronic Hepatitis C ◽  
Mononuclear Cells ◽  
Ldl Receptor ◽  
Surface Expression ◽  
Cell Surface Expression

The LDL receptor (LDL-R) has been proposed as the viral receptor for Hepatitis C virus (HCV). This hypothesis has been based exclusively on in vitro studies. In human mononuclear cells, LDL-R gene expression has been demonstrated to be parallel and be coordinately regulated to gene expression in the human liver. The purpose of the current study was to determine the mononuclear cell surface expression of the LDL receptor in patients with HCV chronic infection according to viral load. Sixty-eight consecutive untreated chronic hepatitis C patients were studied to determine the mononuclear cell surface expression of the LDL-R. LDL-Rs were quantified at the surface of mononuclear cells in fresh blood samples taken after fasting using flow cytometry. LDL-R expression was significantly associated with LDL-cholesterol ( r = −0.25; P = 0.03) and HCV-viral load ( r = 0.37, P = 0.002). In multivariate analysis, the LDL-R expression was significantly associated with HCV viral load, whereas genotype, age, body mass index, and fibrosis were not. In conclusion, our data provided by a human study, suggest that the LDL-R may be one of the receptors implicated in HCV replication.

Identification of a Novel Auto-Antibody Highly Prevalent in Patients with Hepatitis-Associated and Idiopathic Aplastic Anemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3200-3200
Author(s):  
Hiroyuki Takamatsu ◽  
Zhirong Qi ◽  
Tomoyuki Sakurai ◽  
Luis Espinoza ◽  
Naomi Sugimori ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Peptide Mass Fingerprinting ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Healthy Individuals ◽  
Peptide Mass ◽  
Leukemia Cell Lines ◽  
Cd34 Cells

Abstract Abstract 3200 Poster Board III-137 Hepatitis-associated aplastic anemia (HAA) is a subset of acquired AA that is highly responsive to immunosuppressive therapy. The target antigens of the immune system attack in HAA are thought to be a protein shared by both liver and hematopoietic stem cells, since it is usually associated with severe hepatitis of unknown etiology. Screening sera from patients with HAA for the presence of antibodies (Abs) recognizing liver cell-derived proteins may be useful in identifying novel auto-antigens in AA. To test this hypothesis, sera from HAA patients were examined using immunoblotting with a lysate of a hepatocellular carcinoma cell line Huh7 and subsequent peptide mass fingerprinting. Methods and Results The serum of a patient with typical HAA (a 23 year-old male) possessing a small population of paroxysmal nocturnal hemoglobinuria (PNH)-type cells was used for Western blotting (WB) with the lysates of Huh7. A distinct band of 70 kDa protein was revealed. The same band was revealed when the culture supernatant of Huh7 cells was subjected to WB. The peptide mass fingerprinting of the 70 kDa band identified this protein to be heat shock protein (HSP) 72. HSP72 is a stress-inducible protein and extracellular HSP72 enhances the cytotoxicity of CD4+ T cells and NK cells. An examination of the sera from HAA patients, idiopathic acquired AA (IAA) patients and healthy individuals with WB revealed the anti-HSP72 Abs to be detected in 10 of 12 (83%) HAA patients and in 57 of 80 (71%) IAA patients while it was detected only in 8 of 59 (14%) healthy individuals. The prevalence of anti-HSP72 Abs in AA was markedly higher than that of anti-kinectin Abs (39%), anti-PMS1 Abs (10%), anti-DRS-1 Abs (38%) or anti-moesin Abs (37%) reported previously. Anti-HSP72 Abs were frequently detectable both in patients with IAA possessing PNH-type cells (63%) and in patients without PNH-type cells (86%), a finding contrasting to the higher prevalence of anti-DRS-1 Abs and anti-moesin Abs in patients with PNH-type cells than in those without PNH-type cells reported previously. Although anti-HSP72 Abs were detectable in the sera of patients with rheumatoid arthritis and systemic lupus erythematosus, the prevalence was 15% (4 of 27) and 20% (1 of 5), respectively. In contrast to a previous report that detected anti-HSP72 Abs in 24% of patients with chronic hepatitis C, WB failed to detect the Abs in the sera of 4 patients with autoimmune hepatitis and 5 with hepatitis B or C. Ten patients with HAA were treated with immunosuppressive therapy, and 7 of the 8 responders expressed anti-HSP72 Abs. The quantification of the gene expression level of HSP72 by blood cells using real-time PCR demonstrated that the HSP72 mRNA levels were markedly higher in myeloid leukemia cell lines as well as CD34+ cells isolated from 3 healthy individuals in comparison to that in lymphoid or monocytoid leukemia cell lines. HSP72/GAPDH ratios of PBMCs and CD34+ cells from 3 healthy individuals, K562, KH88, OUN-1 were 0.51, 1.31, 1.02, 0.07 and 0.09 respectively. Other leukemia cell lines such as Daudi, Molt-4 and THP-1 did not display detectable levels of HSP72 mRNA. The cell surface expression of HSP72 was examined in various kinds of leukemia cell lines and CD34+ bone marrow (BM) cells derived from 3 healthy individuals using Ab to HSP72 (Clone C92F3A-5) because previous studies demonstrated heat-inducible expression of HSP72 by K562. Flow cytometry detected cell surface HSP72 on immature CML cell lines such as K562 but not on CD34+ BM cells, acute promyelocytic leukemia cell lines such as NB-4 and HL-60, and lymphoid leukemia cell lines such as Molt-4 and Daudi. Exposure to 42°C for 2 h increased the HSP72 expression on K562 cells and Molt-4 cells but not on CD34+ cells. Conclusion Anti-HSP72 Ab is the most prevalent auto-Ab in AA among the auto-Abs previously detected. Given the increased expression of HSP72 by immature myeloid cells as well as stress-inducible cell surface expression of the molecule, immune responses to HSP72 may thus play an essential role in the pathogenesis of HAA and IAA. Disclosures No relevant conflicts of interest to declare.

βig-h3, a Novel Regulator of Adhesion and Differentiation of Human Hematopoietic Stem/Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3640-3640
Author(s):  
Sofieke E Klamer ◽  
Paula B van Hennik ◽  
Daphne C Thijssen-Timmer ◽  
C. Ellen Van der Schoot ◽  
Carlijn Voermans
Keyword(s):  
Cell Surface ◽  
Mrna Expression ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Cell Types ◽  
Cell Surface Expression ◽  
Type I ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 3640 Poster Board III-576 Adult hematopoietic stem cells (HSC) reside in the bone marrow (BM) in so-called niches. Within this specialized microenvironment, the interactions of HSC with adhesion molecules on neighbouring cells and extracellular matrix (ECM) components are thought to be critical for the maintenance of the HSC population. Comparative gene-expression profiling of purified HSC in homeostatic and regenerative conditions allowed the identification of a set of differentially expressed ECM proteins. One of these proteins was the novel ECM protein βg-h3, which plays a role in cell-ECM interactions, by binding to type I, II and IV collagens and cellular integrins. We postulated that βig-h3 could have a role in HSC biology by being both a homeostatic and regenerative regulator of HSC self-renewal and differentiation. First we analyzed the mRNA expression in human CD34+ hematopoietic stem/progenitor cells (HSPC) isolated from BM, mobilized peripheral blood (MPB) and umbilical cord blood (UCB). The expression of βig-h3 was found to be significantly higher in BM-CD34+ cells as compared to MPB-CD34+ cells, suggesting a role for this ECM protein in retaining HSC in the BM. To determine expression of βig-h3 on the various subsets within the heterogeneous CD34+ population, the expression was compared between sorted sub-populations of BM-CD34+ cells: megakaryocyte-erythrocyte-progenitors (MEP: CD38+/CD110+/CD45RA−), common myeloid progenitors (CMP: CD38+/CD110−/CD45RA−), granulocyte-monocyte-progenitors (GMP: CD38+/CD110−/CD45RA+) and more immature CD34+/CD38− HSC. The purity of the sub-populations was analyzed by colony forming assays. These data indicate that at least the mRNA expression of βig-h3 was highest in GMPs. Analysis of different human cell types revealed that the highest βig-h3 mRNA expression is measured in monocytes, dendritic cells and mesenchymal stromal cells (MSC), while its expression in megakaryocytes and HUVEC is comparable to that in HSPC. In addition, cell surface expression of the βig-h3 protein was determined by flowcytometry. βig-h3 was found to be expressed on the cell surface of only a subpopulation of BM derived CD34+ cells (0.5%), monocytes (5%), MSCs (11%) and megakaryocytes (30%). Intracellular flowcytometry staining revealed that βig-h3 is expressed inside CD34+ cells derived from all sources. Since there is evidence in several other cell types that βig-h3 plays a role in enhancing cell adhesion and migration, adhesion experiments using CD34+ cells were performed. These experiments show a significant (p<0.01) two-fold increased adhesion of MPB-CD34+ cells to βig-h3 compared to a BSA coating (mean 40% (SEM ± 9.8%) and 23% (SEM ± 5.0%), respectively, (n=3)). Further experiments showed that adhesion of CD34+ cells to βig-h3 is mediated by both β1- and β2- integrins. The functional relevance of the target proteins in HSC differentiation and self-renewal was studied by lentiviral mediated overexpression. We used a βig-h3-SIN-GFP vector or a control SIN-GFP vector to transduce CD34+ cells isolated from MPB or UCB and cultured them towards a megakaryocytic lineage using TPO, SCF, Flt3 and IL6. Overexpression of βig-h3 in MPB and UCB-CD34+ cells resulted in an acceleration of the megakaryopoiesis and in an increased percentage of mature megakaryocytic cells (i.e. CD41+) two weeks after transduction. In conclusion, βig-h3 is an adhesive protein for HSPCs and GMP's express significantly more βig-h3 as compared to other CD34+ subsets. Moreover, ectopic expression of βig-h3 in CD34+ cells accelerates differentiation towards megakaryocytes. These data suggest that upregulation of βig-h3 in HSCs may be a vital element driving lineage commitment of HSCs in homeostatic or regenerative conditions. Disclosures: No relevant conflicts of interest to declare.

Erlotinib Inhibits ABC Transporters of AML Progenitors with Stem Cell Features and Increases Chemosensitivity to Current AML Drugs

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2163-2163
Author(s):  
Marie Sebert ◽  
Elodie Lainey ◽  
Sylvain Thepot ◽  
Maximilien Tailler ◽  
Lionel Ades ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Surface ◽  
Abc Transporters ◽  
Ex Vivo ◽  
Myeloid Cell ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Drug Efflux ◽  
Induced Apoptosis ◽  
Over Time

Abstract Abstract 2163 Background: Treatment failure in AML is attributed to the persistence of AML progenitors able, among others, to efflux chemotherapeutic drugs via ABC-transporters. Increased efflux capacity is considered a stem cell feature, and therapeutic inhibition may increase chemosensitivity and help eradicate this progenitor population. Nevertheless, clinical studies assessing a potential benefit of ABC-inhibitors in AML treatment showed no significant survival advantage, possibly because AML cells express different ABC-transporters and classical inhibitors target only a restricted type of efflux channels. We assessed the efficacy of the TKI erlotinib (Erlo) to antagonize drug efflux via most important AML-associated efflux channels, ie P-gp, MRP and BCRP. Methods: Overall drug efflux via ABC-transporters (substrate: mitoxantrone-MTZ), and specific efflux via P-gp (substrates: DioC23 and rhodamine-123), MRP (substrates: calcein and CDCFDA) and BCRP (substrate: Hoechst 33342) were quantified by FACS at 1h and 6h following incubation with 10mM Erlo. Biochemical inhibitors of the respective ABC-transporters (CSA, verapamil, MK-571, KO143) served as controls. Surface expression of P-gp, MRP and BCRP was quantified by FACS. To assess chemosensitivity, 10mM Erlo was combined to AraC (100nM), doxorubicine (Dox, 100nM), or VP-16 (1mM) and apoptosis over-time (24, 48, 72h) quantified by DioC3(6)/PI staining. Assays were carried out in myeloid cell lines (KG-1, MOLM-13, HL-60) and ex vivo AML cells (n=3). Immaturity of AML cells was determined in 2 samples by comparing CD34+ versus CD34- cells, and in one pt by co-staining for CD34, CD38, CD123 and CD133. Results: We found that I) Erlo inhibited efflux via P-gp and MRP as demonstrated by increased intracellular retention of DioC23/Rho-123, and calcein/CDCFDA, respectively; II) this degree of inhibition was higher in KG-1 cells than in MOLM-13 or HL-60 cells; III) inhibition of drug efflux was observed already at 1h of incubation, increased over time (6h); IV) Erlo increased intracellular retention of MTZ faster (at 1h with a further increase at 6h) and at least to the same extent than a combination of all three biochemical efflux inhibitors, showing that Erlo's capacity to hinder drug efflux is not restricted to a single ABC-transporter: V) surface expression of P-gp, MRP and BCRP was strongest on KG-1 cells and not altered upon 1h and 6h of Erlo incubation VI) Erlo increased Dox- and VP16-induced apoptosis (48h KG-1: Erlo alone 20%, Dox alone 10%, VP-16 alone 20%, Erlo+Dox: 40%, VP-16+Erlo: 70%), while having no impact on AraC-induced apoptosis; VI) this pattern of chemosensitization was observed in all myeloid cell lines, but once more most pronounced in KG-1 cells. To test the hypothesis that Erlo has comparable effects in pt-derived AML cells ex vivo, we showed by concomitant cell surface staining that I) immature AML subpopulations had a higher efflux capacity (notably via P-gp) than their more mature counterparts (i.e. in one pt with chemoresistant AML: DioC23/Rho-123 fluorescence twice as high in the CD34-/CD38+, CD123+, CD133- than in the CD34+/CD38dim, CD123-, CD133+ subpopulation); II) cell surface expression of P-gp is twice as high in this more immature population (CD34+/CD38dim, CD123-, CD133+) than in CD34-/CD38+, CD123+, CD133+ cells; III) Erlo antagonizes drug efflux via P-gp and MRP at 1h (increasing further at 6h) of incubation; IV) this effect is most pronounced in the immature progenitor cells (1h: decrease of DioC23/Rho-123 efflux in CD34-/CD38+, CD123+, CD133- cells by about 50% and in the more immature CD34-/CD38+, CD123-, CD133+ cells by about 70%); V) Erlo diminishes cell surface expression of P-gp (48h), most effectively in the progenitor populations (by 30% in the CD34-/CD38+, CD123+, CD133- cells versus 50% in CD34-/CD38+, CD123+, CD133- cells); VI) Erlo is able to retain MTZ in both CD34- and CD34+ AML-subpopulations; VII) these effects are accompanied by an increased sensitivity towards Dox and VP-16; VIII) Erlo-induced chemosensitization is higher in the CD34+ than in CD34- AML cells. Conclusions: We here provide novel evidence that erlotinib is able to overcome the stem cell features of increased expression and functionality of ABC-transporters thereby antagonizing the intrinsic chemoresistance of (immature) AML cells. Those results suggest a potential clinical interest of combining erlotinib to chemotherapy in AML Disclosures: Fenaux: CELGENE, JANSSEN CILAG, AMGEN, ROCHE, GSK, NOVARTIS, MERCK, CEPHALON: Consultancy.

CD81-Dependent Trafficking of CD19: Restoration of CD19 Surface Expression in Human B Cells Harboring A CD81 Mutation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1049-1049
Author(s):  
Shoshana Levy ◽  
Chiung-Chi Kuo ◽  
Yael Sagi ◽  
Homer Chen ◽  
Neta Kela-Madar ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Surface ◽  
B Cell ◽  
Cell Line ◽  
Conflicts Of Interest ◽  
Surface Expression ◽  
Antibody Deficiency ◽  
Cell Surface Expression ◽  
Dominant Negative Effect ◽  
Proximity Ligation Assay

Abstract Abstract 1049 Introduction: A 6-year-old girl, who was diagnosed with a primary antibody deficiency, had B cells lacking surface CD19. However, both her CD19 alleles were normal and the impairment was actually caused by a homozygous exon splice site mutation in CD81 (1). The patient's B cells also lacked surface CD81 and produced an immature glycosylated CD19 protein that was retained intracellularly. Interestingly, this human deficiency differed from that of CD81 knockout mice as the latter still express a low level of CD19 on their B cells. Methods: We used an EBV-transformed B cell line from this patient to better understand i) the difference between the human and mouse CD81 deficiencies and ii) how CD81 controls the trafficking of CD19 to the cell surface. We reasoned that the truncated human CD81 mutant (CD81mut) protein might be expressed intracellularly. Indeed, whereas most anti-CD81 mAbs did not recognize CD81mut, we identified one that bound the mutated form and used it in this study. We also expressed the human CD81mut in a CD81-deficient mouse B cell line to determine if it could negatively regulate CD19 surface expression. Results: We show that the CD81mut protein is indeed expressed intracellularly in the patient's EBV-transformed B cells. We then used a proximity ligation assay to demonstrate that the truncated CD81mut protein interacts intracellularly with CD19. However, this interaction with the CD81mut protein abrogated carbohydrate maturation and the trafficking of CD19 to cell surface. We therefore expressed the CD81mut in CD81KO mouse B cells, which still express low levels of surface CD19, and found that it did not exert a dominant negative effect on CD19 surface expression. Finally, we used this reconstitution system to identify specific CD81 domains that restored carbohydrate maturation and cell surface expression of the CD19 molecule in the patient's B cells. Conclusion: This specific case of antibody deficiency was manifested because of lack of surface expression of CD19, an important B cell signaling molecule. However, the maturation of CD19 and its trafficking to the cell surface require the presence of specific domains of the tetraspanin CD81 molecule. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Aminooxypentane-RANTES Induces CCR5 Internalization but Inhibits Recycling: A Novel Inhibitory Mechanism of HIV Infectivity

1998 ◽  
Vol 187 (8) ◽  
pp. 1215-1224 ◽  
Author(s):  
Matthias Mack ◽  
Bruno Luckow ◽  
Peter J. Nelson ◽  
Josef Cihak ◽  
Graham Simmons ◽  
...  
Keyword(s):  
Cell Surface ◽  
Hiv Infection ◽  
Mononuclear Cells ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Receptor Internalization ◽  
Cell Surface Expression ◽  
Macrophage Infection ◽  
Potent Inhibition ◽  
Hiv 1

CCR5, a chemokine receptor expressed on T cells and macrophages, is the principal coreceptor for M-tropic HIV-1 strains. Recently, we described an NH2-terminal modification of the CCR5 ligand regulated on activation, normal T cell expressed and secreted (RANTES), aminooxypentane-RANTES (AOP-RANTES), that showed potent inhibition of macrophage infection by HIV-1 under conditions where RANTES was barely effective. To investigate the mechanism of AOP-RANTES inhibition of HIV infectivity we examined the surface expression of CCR5 using a monoclonal anti-CCR5 antibody, MC-1. We demonstrate that AOP-RANTES rapidly caused &gt;90% decrease in cell surface expression of CCR5 on lymphocytes, monocytes/ macrophages, and CCR5 transfected Chinese hamster ovary (CHO) cells. RANTES also caused a loss of cell surface CCR5, although its effect was less than with AOP-RANTES. Significantly, AOP-RANTES inhibited recycling of internalized CCR5 to the cell surface, whereas RANTES did not. When peripheral blood mononuclear cells are cultured for prolonged periods of time in the presence of RANTES, CCR5 expression is comparable to that seen on cells treated with control medium, whereas there is no CCR5 surface expression on cells cultured in the presence of AOP-RANTES. Immunofluorescence indicated that both AOP-RANTES and RANTES induced downmodulation of cell surface CCR5, and that the receptor was redistributed into endocytic organelles containing the transferrin receptor. When RANTES was removed, the internalized receptor was recycled to the cell surface; however, the receptor internalized in the presence of AOP-RANTES was retained in endosomes. Using human osteosarcoma (GHOST) 34/CCR5 cells, the potency of AOP-RANTES and RANTES to inhibit infection by the M-tropic HIV-1 strain, SF 162, correlated with the degree of downregulation of CCR5 induced by the two chemokines. These differences between AOP-RANTES and RANTES in their effect on receptor downregulation and recycling suggest a mechanism for the potent inhibition of HIV infection by AOP-RANTES. Moreover, these results support the notion that receptor internalization and inhibition of receptor recycling present new targets for therapeutic agents to prevent HIV infection.

Enolase-1 promotes plasminogen-mediated recruitment of monocytes to the acutely inflamed lung

Blood ◽  
2009 ◽  
Vol 113 (22) ◽  
pp. 5588-5598 ◽  
Author(s):  
Malgorzata Wygrecka ◽  
Leigh M. Marsh ◽  
Rory E. Morty ◽  
Ingrid Henneke ◽  
Andreas Guenther ◽  
...  
Keyword(s):  
Cell Surface ◽  
Binding Site ◽  
Mononuclear Cells ◽  
Surface Expression ◽  
Mononuclear Phagocytes ◽  
Cell Surface Expression ◽  
U937 Cells ◽  
Blood Monocytes ◽  
Monocyte Migration

Abstract Cell surface–associated proteolysis plays a crucial role in the migration of mononuclear phagocytes to sites of inflammation. The glycolytic enzyme enolase-1 (ENO-1) binds plasminogen at the cell surface, enhancing local plasmin production. This study addressed the role played by ENO-1 in lipopolysaccharide (LPS)–driven chemokine-directed monocyte migration and matrix invasion in vitro, as well as recruitment of monocytes to the alveolar compartment in vivo. LPS rapidly up-regulated ENO-1 cell-surface expression on human blood monocytes and U937 cells due to protein translocation from cytosolic pools, which increased plasmin generation, enhanced monocyte migration through epithelial monolayers, and promoted matrix degradation. These effects were abrogated by antibodies directed against the plasminogen binding site of ENO-1. Overexpression of ENO-1 in U937 cells increased their migratory and matrix-penetrating capacity, which was suppressed by overexpression of a truncated ENO-1 variant lacking the plasminogen binding site (ENO-1ΔPLG). In vivo, intratracheal LPS application in mice promoted alveolar recruitment of monocytic cells that overexpressed ENO-1, but not of cells overexpressing ENO-1ΔPLG. Consistent with these data, pneumonia-patients exhibited increased ENO-1 cell-surface expression on blood monocytes and intense ENO-1 staining of mononuclear cells in the alveolar space. These data suggest an important mechanism of inflammatory cell invasion mediated by increased cell-surface expression of ENO-1.

scholarly journals CD44-Mediated Adhesiveness of Human Hematopoietic Progenitors to Hyaluronan Is Modulated by Cytokines

Blood ◽  
1997 ◽  
Vol 89 (6) ◽  
pp. 1905-1914 ◽  
Author(s):  
Stéphane Legras ◽  
Jean-Pierre Lévesque ◽  
Rachida Charrad ◽  
Kohji Morimoto ◽  
Caroline Le Bousse ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Adhesive Interaction ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Marrow Stroma ◽  
Adhesive Interactions

Abstract Adhesive interactions between CD34+ hematopoietic progenitor cells (HPC) and bone marrow stroma are crucial for normal hematopoiesis, yet their molecular bases are still poorly elucidated. We have investigated whether cell surface proteoglycan CD44 can mediate adhesion of human CD34+ HPC to immobilized hyaluronan (HA), an abundant glycosaminoglycan of the bone marrow extracellular matrix. Our data show that, although CD34+ cells strongly express CD44, only 13.3% ± 1.1% spontaneously adheres to HA. Short-term methylcellulose assay showed that HA-adherent CD34+ cells comprised granulo-monocytic and erythroid committed progenitors (19.6% ± 2.5% and 7.3% ± 1.0% of the input, respectively). More primitive progenitors, such as pre–colony-forming units, also adhered to HA. Moreover, we found that CD44-mediated adhesion of CD34+ cells to HA could be enhanced by phorbol 12-myristate 13-acetate (PMA), the function-activating anti-CD44 monoclonal antibody H90, and cytokines such as granulocyte-monocyte colony-stimulating factor, interleukin-3 (IL-3), and stem cell factor. Enhancement through PMA required several hours, was protein-synthesis–dependent, and was associated with an increase of CD44 cell surface expression, whereas stimulation of adhesion by H90 monoclonal antibody and cytokines was very rapid and without alteration of CD44 expression. H90-induced activation occurred at 4°C and lasted for at least 2 hours, whereas activation by cytokines required incubation at 37°C and was transient. These data, which show for the first time that CD34+ HPC can directly adhere to HA via CD44, point out that this adhesive interaction to HA is a process that may also be physiologically regulated by cytokines.

scholarly journals The rs35112940 CD33 Polymorphism Reduces CD33 Internalization and Efficacy of CD33-Directed Gemtuzumab Ozogamicin

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2247-2247
Author(s):  
Mohammed O Gbadamosi ◽  
Vivek M. Shastri ◽  
Soheil Meshinchi ◽  
Jatinder K. Lamba
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Conflicts Of Interest ◽  
Surface Expression ◽  
Gemtuzumab Ozogamicin ◽  
Cell Surface Expression ◽  
Specific Cell ◽  
Antibody Drug Conjugate ◽  
Critical Feature ◽  
The Impact

Abstract Background CD33 is a myeloid-specific cell surface protein widely expressed on acute myeloid leukemia (AML) cells making it an excellent immunotherapeutic target. Current CD33-directed immunotherapeutic treatment strategies include gemtuzumab ozogamicin (GO), an antibody-drug conjugate (ADC) which was approved for the treatment AML in 2017 and has demonstrated promising results thus far. The mechanism of action of GO begins with recognition of CD33 by the antibody portion of GO, followed by internalization of the CD33-GO complex, and finally delivery of free calicheamicin molecules to the cell to induce cellular apoptosis. As such, modifications that impact these steps on any level presumably impact the response and overall efficacy of GO. Indeed, previous studies from our group have identified germline variations in CD33 that are associated with differences in CD33 structure, CD33 cell surface expression levels, and clinical outcomes in response to GO. Among these germline variations is rs35112940 (G&gt;A; Arg304Gly), a missense polymorphism which is located in exon five of CD33 adjacent to the cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) domain, a critical feature for CD33 internalization. While our previous work identified statistical associations between the A allele of rs35112940 and lower CD33 expression and reduced benefit from treatment using GO, these results are yet to be validated functionally. Additionally, it still remains unknown if the impact of the rs35112940 variation is due to reduced CD33 expression alone or if the rs35112940 variation also impacts CD33 internalization thereby modulating CD33 efficacy. Methods To functionally validate the effect of the rs35112940 variant, we used CRISPR/cas9 to knockout CD33 in HL60 cells and subsequently engineered the HL60-CD33 KO cells to express either wildtype CD33 (HL60-CD33 FL) or CD33 encoding the rs35112940 variant (HL60-CD33 FL-rs35112940). The engineered cells were then treated with GO for 48 hours to capture the impact of the rs35112940 variation on the efficacy of GO. To assess the impact of the rs35112940 variation on CD33 internalization, we performed a flow cytometry-based internalization assay using secondary antibodies to capture the remaining amounts of CD33 present on the cell surface after 4 hours allowing us to determine the internalization of CD33 over time. Results All engineered cells expressed CD33 with less than 1-log fold difference in median fluorescence intensity (MFI) (HL60-CD33 FL MFI vs HL60-CD33 FL-rs35112940 MFI: 22536 vs 24882, Figure 1) and thus we were able to characterize the impact of the rs35112940 variant independent of its impact on CD33 cell surface expression. After 48-hour treatment with 250 ng/mL of GO, we observed that HL60-CD33 FL-rs35112940 cells were more resistant to GO than HL60-CD33 FL cells (66.4% vs 46.5% cell viability, P = 0.02, Figure 2A). Similar results were observed at multiple concentrations of GO. Given the proximity of the rs35112940 loci to the ITIM domain of CD33, we hypothesized that the rs35112940 variation may impact CD33 internalization as well. In a flow cytometry-based internalization assay over a 4-hour window, we observed that that HL60-CD33 FL-rs35112940 cells had an approximate 10% reduction in CD33 internalization in comparison to HL60-CD33 FL cells (Figure 2B). Taken together these results provide insight into the effect of the rs35112940 variant on GO efficacy and CD33 biology, corroborating our previous findings, and support the use of CD33 polymorphisms to guide patient selection for treatment with GO. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Human CD34+CXCR4− sorted cells harbor intracellular CXCR4, which can be functionally expressed and provide NOD/SCID repopulation

Blood ◽  
2002 ◽  
Vol 100 (8) ◽  
pp. 2778-2786 ◽  
Author(s):  
Orit Kollet ◽  
Isabelle Petit ◽  
Joy Kahn ◽  
Sarit Samira ◽  
Ayelet Dar ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Surface ◽  
Cord Blood ◽  
Surface Expression ◽  
Scid Mice ◽  
Cell Surface Expression ◽  
Human Cord Blood ◽  
Murine Bone Marrow ◽  
Human Cd34 ◽  
Cd34 Cells

Homing and repopulation of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice by enriched human CD34+stem cells from cord blood, bone marrow, or mobilized peripheral blood are dependent on stromal cell-derived factor 1 (SDF-1)/CXCR4 interactions. Recently, human cord and fetal blood CD34+CD38−CXCR4− and CXCR4+ cells, sorted with neutralizing anti-CXCR4 monoclonal antibody (mAb), were shown to have similar NOD/SCID repopulation potential. Herein we report that human cord blood CD34+CXCR4+ (R4+) and CD34+CXCR4− (R4−) subsets, sorted with neutralizing anti-CXCR4 mAb, engrafted NOD/SCID mice with significantly lower levels of human cells compared with nonsorted and SDF-1–migrated CD34+ cells. Coinjection of purified cells with 10 μg anti-CXCR4 mAb significantly reduced engraftment of all CD34+ subsets, and 50 μg completely abrogated engraftment by R4− and CD34+ cells. Importantly, R4− cells harbor intracellular CXCR4, which can be rapidly induced to cell surface expression within a few hours. Moreover, 48 hours of cytokine stimulation resulted in up-regulation of both cell surface and intracellular CXCR4, restoring migration capacities toward a gradient of SDF-1 and high-level NOD/SCID repopulation potential. In addition, homing of sorted R4− cells into the murine bone marrow and spleen was significantly slower and reduced compared to CD34+ cells but yet CXCR4 dependent. In conclusion, R4− cells express intracellular CXCR4, which can be functionally expressed on the cell membrane to mediate SDF-1–dependent homing and repopulation. Our results suggest dynamic CXCR4 expression on CD34+ stem and progenitor cells, regulating their motility and repopulation capacities.

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