Lysates Of Granulocytes Research Articles

Molecular and functional characterization of porcine Siglec-3/CD33 and analysis of its expression in blood and tissues

A cDNA clone encoding a 380 a-a type 1 transmembrane protein with homology to human Siglec-3/CD33 was obtained from a swine small intestine library. An analysis of protein sequence identified two immunoglobulin-like domains, a transmembrane region, and a carboxi-terminal tail with two tyrosine-based signalling motifs. Binding assays of Siglec-3 transfected CHO cells to polyacrylamide glycoconjugates showed a preference for α2–6-linked sialic acids. Using mAbs raised against a fragment containing the two Ig-like domains, porcine Siglec-3 was found to be expressed on monocytes and granulocytes, and their bone marrow precursors. It was also detected in lymph node, splenic and alveolar macrophages. MAbs immunoprecipitated, from granulocyte lysates, a protein of 51–60 kDa under both non-reducing and reducing conditions. MAbs were also used to analyse functional activity of Siglec-3 on bone marrow and blood cells. Engagement of Siglec-3 by mAb had no apparent effect on cell proliferation or cytokine production.

Molecular characterization and expression of porcine Siglec-5

In this study we describe the characterization of the porcine orthologue of Siglec-5. A cDNa clone was obtained from a porcine cDNa library derived from swine small intestine which encodes a 555 a-a type 1 transmembrane protein with sequence homology to human Siglec-5. This protein consists of four Ig-like domains, a transmembrane region, and a cytoplasmic tail with two tyrosine-based signalling motifs. When expressed as a recombinant protein fused to the Fc region of human IgG1, porcine Siglec-5 was able to bind porcine red blood cells in a sialic acid-dependent manner. Monoclonal antibodies (mAb) were developed against porcine Siglec-5 and used to analyse its expression in bone marrow and blood cells, and lymphoid tissues. Porcine Siglec-5 expression was mainly restricted to myelomonocytic cells and their precursors, being detected also, although at low levels, on plasmacytoid dendritic cells and B lymphocytes. In lymphoid tissues, ellipsoids of the spleen and subcapsular and medullar sinuses of lymph nodes were positive for Siglec-5. These mAbs were able to precipitate, from granulocyte lysates, a protein of approximately 85kDa under non-reducing conditions, indicating that porcine Siglec-5 is expressed as a monomer in the plasma membrane.

Myeloid Derived Suppressor Cells (MDSCs) Are Increased and Exert Immunosuppressive Activity In CML Patients At Diagnosis

IntroductionIn some solid tumors it has been demonstrated that a subpopulation of myeloid cells, defined as “myeloid-derived suppressor cells” (MDSCs), plays an important role in inducing T cell tolerance by production of arginase 1 (arg1) that depletes microenvironment of arginine, an essential aminoacid for T cell function. Since chronic myeloid leukemia (CML) patients have high levels of immature myeloid cells it is of interest to investigate if these cells have MDSCs phenotype and activity. The aim of this study was to analyze MDSCs and investigate their activity in CML patients. MethodsMDSCs were analyzed in peripheral blood (PB) of 20 healthy donors (HD) and 30 CML patients at diagnosis. In 21 patients MDSCs were also measured during TKI treatment. Granulocytic MDSCs (G-MDSCs) were identified as CD11b+CD33+CD14-HLADR- cells, while the monocytic MDSCs (Mo-MDSCs) as CD14+HLADR by cytofluorimetric analysis. Arg1 expression was assessed using real time PCR and Western Blot. Arg activity was measured in granulocyte lysates using a colorimetric test after enzymatic activation and arginine hydrolysis. Microvesicles (MV) were isolated from CML serum at diagnosis (n=5) by sequential ultracentrifugation. ResultsCML patients showed high levels of Mo- and G-MDSCs at diagnosis in comparison to HD (41±8 and 82,5±12,2% respectively for CML vs 9±2,1 and 55±5,3% for HD; p<0.001), while after TKIs therapy both subpopulations decreased, returning to normal values. T-reg (CD4+ CD25high Foxp3+ cells) were also significantly increased in CML patients at diagnosis in respect to HD (9±2% vs 6,1±0,8%, p<0.001) with a significant correlation with the percentage of Gr-MDSCs (r=0,6254; p<0.001). Both in PB and purified granulocytic cells, Arg1 expression showed a 30 fold increase in CML at diagnosis compared to HD (p<0.001) and decreased after therapy. The same data were confirmed by Western Blot analysis. Arg enzymatic activity in granulocytes resulted also increased in CML (n=10) compared to HD (n=10) (p<0.001). The suppressive function of CML G-MDSCs was demonstrated by their ability to inhibit the proliferation of CFSE+ HD T cells (p<0.001). In addition, an increase of Mo-MDSCs in vitro was observed after incubation of HD monocytes with CML sera (29±13%; p<0.0001) or MV (8±2,8%; p<0.05). ConclusionsMDSCs are increased in CML patients at diagnosis and decrease during TKIs treatment. CML granulocytes have high arg1 activity and immunosuppressive activity. Moreover, CML serum as well as CML microvesicles increase the percentage of HD Mo-MDSCs. Disclosures:No relevant conflicts of interest to declare.

Myeloid-Derived Suppressor Cells Increase in Chronic Myeloid Leukemia and Exert Immune Suppressive Activity.

AbstractAbstract 2779 Background:Tumor cells are able to develop immune evasion mechanisms which induce a state of immune tolerance and inactivate tumor-specific T cells. In this context, in some solid tumors it has been demonstrated that a subpopulation of myeloid cells, defined as “myeloid-derived suppressor cells” (MDSCs), plays an important role in inducing T cell tolerance by production of arginase that depletes microenvironment of arginine, an essential aminoacid for T cell function. Since chronic myeloid leukemia (CML) patients have high levels of immature myeloid cells it is of interest to investigate if these cells have MDSCs phenotype and activity. Aim:The aim of this study was to analyze MDSCs and investigate their involvement in T-cell anergy of CML patients. Methods:MDSCs were analyzed in peripheral blood (PB) of 13 CML patients (at diagnosis and during therapy) and healthy donors (HD; n=20) by cytofluorimetric analysis (CD14+DR- for monocytic MDSCs and CD11b+CD33+CD14-DR- for granulocytic MDSCs). Arginase 1 expression was assessed in PB of HD and CML patient using real time PCR. Purification of granulocytes, monocytes and lymphocytes from PB was performed by a positive magnetic separation kit (EasySep, STEMCELL Technologies). Arginase activity was measured in granulocyte lysates using a colorimetric test after enzymatic activation and arginine hydrolysis. To evaluate the activation of CD3+ T lymphocytes after incubation with phytoemagglutinin, we analyzed at 24, 48, 72 h the following markers: CD69+, CD71+, DR+. Microvesicles were isolated from CML serum at diagnosis (n=5) by sequential ultracentrifugation. Results:CML patients showed high levels of monocytic and granulocytic MDSCs at diagnosis in comparison to HD (63±8 and 83±12,2% respectively in CML vs 4,9±2,1 and 55,8±5,3% respectively in HD; p<0.001) while after 3–6 months of tyrosine kinase inhibitors (TKIs) therapy MDSC levels returned to normal values. Either in PB and in the purified granulocytes subpopulation, arginase1 expression showed a 30 fold increase in CML at diagnosis (CML vs HD: p<0.01) and decreased after therapy. We also evaluated arginase enzymatic activity in granulocytes and we found it increased in CML patients (n=4) compared to HD (n=5) (p<0.05). CML as well as HD T lymphocytes showed a normal activation in vitro which was significantly lost when they was incubated with CML serum (n=4). In addition, an increase of monocytic MDSCs in vitro was observed after incubation of HD monocytes with CML serum (39±6%; p<0.01) or microvescicles (9,2±1,2%; p<0.05) compared to control serum. Conclusions:Granulocytic and monocytic MDSCs are increased in CML patients at diagnosis and decrease during TKIs treatment. Their levels also correlates with Arginase 1 expression and enzymatic activity in granulocytes. CML serum as well as CML microvesicles increase the percentage of HD monocytic MDSCs. Moreover, CML serum leads to anergy of T lymphocytes, probably by Arginase 1 secretion. Disclosures:Off Label Use: Eltrombopag is a thrombopoietin receptor agonist indicated for the treatment of thrombocytopenia in patients with chronic immune (idiopathic) thrombocytopenic purpura (ITP).

Leucocytes are a major source of circulating nicotinamide phosphoribosyltransferase (NAMPT)/pre-B cell colony (PBEF)/visfatin linking obesity and inflammation in humans

Aims/hypothesisNicotinamide phosphoribosyltransferase (NAMPT) is a multifunctional protein potentially involved in obesity and glucose metabolism. We systematically studied the association between circulating NAMPT, obesity, interventions and glucose metabolism and investigated potential underlying inflammatory mechanisms.MethodsFasting morning NAMPT serum levels were measured in cohorts of lean vs obese children, cohorts of intervention by lifestyle, exercise and bariatric surgery, and during an OGTT. In addition, mRNA expression, protein production and enzymatic activity of NAMPT were assessed from isolated leucocytes and subpopulations.ResultsCirculating NAMPT was significantly elevated in obese compared with lean children and declined after obesity interventions concomitantly with the decline in BMI, high-sensitivity C-reactive protein (hsCrP) and leucocyte counts. Circulating NAMPT significantly correlated with glucose metabolism and cardiovascular variables in univariate analyses, but only the association with glucose response during an OGTT was independent from BMI. We therefore assessed the NAMPT dynamic following an oral glucose load and found a significant decline of NAMPT levels to 77.0 ± 0.1% as a function of time, and insulin-to-glucose ratio during an OGTT in obese insulin-resistant adolescents. Circulating NAMPT was, however, most strongly associated with leucocyte counts (r = 0.46, p < 0.001). The leucocyte count itself determined significantly and independently from BMI insulin resistance in multiple regression analyses. We systematically evaluated NAMPT expression among several tissues and found that NAMPT was predominantly expressed in leucocytes. In subsequent analyses of leucocyte subpopulations, we identified higher NAMPT protein concentrations in lysates of granulocytes and monocytes compared with lymphocytes, whereas granulocytes secreted highest amounts of NAMPT protein into cell culture supernatant fractions. We confirmed nicotinamide mononucleotide enzymatic activity of NAMPT in all lysates and supernatant fractions. In monocytes, NAMPT release was significantly stimulated by lipopolysaccharide (LPS) exposure.ConclusionsLeucocytes are a major source of enzymatically active NAMPT, which may serve as a biomarker or even mediator linking obesity, inflammation and insulin resistance.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-010-2042-z) contains supplementary material, which is available to authorised users.

Partial characterization of feline myeloperoxidase and investigation of its potential role as an autoantigen in hyperthyroid cats

To partially characterize the cDNA, amino acid sequence, and tertiary structure of feline myeloperoxidase, describe its cellular location in mature granulocytes, and determine whether hyperthyroid cats have anti-myeloperoxidase antibody. Bone marrow RNA and whole blood from cats of various sources and feline serum samples submitted for measurement of total thyroxine concentration from September 2006 to July 2007. Feline myeloperoxidase cDNA was amplified from bone marrow RNA; presumptive splice sites were determined by comparison with human sequences. Intracellular localization of myeloperoxidase in granulocytes was determined by use of immunofluorescence and electron microscopy, and molecular weight and partial tertiary structure were determined by use of immunoblotting of granulocyte lysates. Anti-human myeloperoxidase (hMPO) antibody was detected via ELISA. A 2,493-bp sequence encompassing the 2,160-bp cDNA with presumably the same number and size of exons as hMPO was generated. Translation predicted 85% homology with hMPO. Feline myeloperoxidase was localized to neutrophil primary granules, and immunoblotting revealed heavy and light bands with molecular weights similar to those of hMPO. The prevalence of anti-hMPO antibody did not differ between nonhyperthyroid and hyperthyroid cats or among hyperthyroid cats subclassified by treatment modality. Moderate homology existed between feline myeloperoxidase and hMPO cDNA and protein. Although findings suggested a similar tertiary structure and function for the 2 proteins, they also suggested that inability to detect a high prevalence of anti-hMPO antibody in hyperthyroid cats may be attributable to antigenic differences between the human and feline proteins rather than a lack of autoantibody.

Evolution and Function of Leukocyte RNase A Ribonucleases of the Avian Species, Gallus gallus

In this study, we explore the evolution and function of two closely related RNase A ribonucleases from the chicken, Gallus gallus. Separated by approximately 10 kb on chromosome 6, the coding sequences of RNases A-1 and A-2 are diverging under positive selection pressure (dN > dS) but remain similar to one another (81% amino acid identity) and to the mammalian angiogenins. Immunoreactive RNases A-1 and A-2 (both approximately 16 kDa) were detected in peripheral blood granulocytes and bone marrow. Recombinant proteins are ribonucleolytically active (kcat = 2.6 and 0.056 s(-1), respectively), and surprisingly, both interact with human placental ribonuclease inhibitor. RNase A-2, the more cationic (pI 11.0), is both angiogenic and bactericidal; RNase A-1 (pI 10.2) has neither activity. We demonstrated via point mutation of the catalytic His110 that ablation of ribonuclease activity has no impact on the bactericidal activity of RNase A-2. We determined that the divergent domains II (amino acids 71-76) and III (amino acids 89-104) of RNase A-2 are both important for bactericidal activity. Furthermore, we demonstrated that these cationic domains can function as independent bactericidal peptides without the tertiary structure imposed by the RNase A backbone. These results suggest that ribonucleolytic activity may not be a crucial constraint limiting the ongoing evolution of this gene family and that the ribonuclease backbone may be merely serving as a scaffold to support the evolution of novel, nonribonucleolytic proteins.

Identification of the leukocyte adhesion molecules CD11 and CD18 as receptors for type 1-fimbriated (mannose-specific) Escherichia coli

Attachment of bacteria to phagocytic cells may be mediated by lectin-carbohydrate interactions, resulting in lectinophagocytosis. The best-studied system is the interaction of type 1-fimbriated (mannose-specific) Escherichia coli with human phagocytic cells. Here we demonstrate that the leukocyte integrins CD11 and CD18 (CD11/CD18) constitute the major receptors for type 1-fimbriated E. coli. Bacteria were bound in a dose-dependent and saturable manner to CD11/CD18, which was immobilized to microwells, whereas nonfimbriated E. coli cells failed to bind. The binding was efficiently inhibited (82 to 85%) by methyl-alpha-mannoside but not by galactose, and it was reduced by treatment of the immobilized CD11/CD18 with sodium metaperiodate, endoglycosidase H, or a mixture of endoglycosidase F and N-glycosidase. The fimbriated bacteria also bound to CD11a,b,c and CD18 separated by polyacrylamide gel electrophoresis with sodium dodecyl sulfate and blotted onto nitrocellulose paper. This binding was inhibited specifically by methyl-alpha-mannoside and was significantly diminished by treatment of the blots with sodium metaperiodate. Only minimal binding to the blotted CD11/CD18 that had been deglycosylated enzymatically prior to electrophoresis was observed. On blots of granulocyte lysates, specific binding to two glycoproteins (Mrs, 90,000 to 100,000 and 165,000) with mobilities similar to that of CD11/CD18 was observed. Monoclonal antibodies to CD11a, CD11b, or CD18 inhibited the binding of the bacteria to intact human granulocytes by 55 to 80%, whereas antibodies against other leukocyte surface antigens were not inhibitory. We conclude that type 1-fimbriated E. coli binds to human granulocytes via the oligomannose and hybrid N-linked units of CD11/CD18. Since CD11b/CD18 and CD11c/CD18 are known to serve as receptors for complement fragment iC3b, this study provides a link between opsonophagocytosis and lectinophagocytosis.

Endothelial cell-derived high molecular weight von Willebrand factor is converted into the plasma multimer pattern by granulocyte proteases

We have previously found that the von Willebrand factor released by cultured human umbilical vein endothelial cells appeared as a single high molecular weight band in glyoxyl agarose electrophoresis. In the present studies we report that this high molecular weight endothelial cell-derived von Willebrand factor, when incubated with granulocyte lysates, was cleaved into a series of multimers indistinguishable from those seen in normal plasma (or type II von Willebrand disease). This von Willebrand factor-cleaving activity was released from granulocytes by calcium ionophore A23187 but was not detected in cytosolic fractions depleted of granular contents. It was inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride. This von Willebrand factor-cleaving activity thus provides a possible mechanism for the generation of plasma von Willebrand factor multimers from the high molecular weight form of von Willebrand factor secreted by endothelial cells.

Purification of functional RNA from human granulocytes.

The presence, in granulocytes, of high levels of nuclease activity makes it difficult to isolate intact RNA from these cells. We have developed a method that allows purification of functional RNA from normal granulocytes as determined by capability for reverse transcription and in vitro translation. We have shown that a considerable amount of ribonuclease activity remains in granulocyte lysates, even after the addition of heparin or vanadyl ribonucleoside complexes. RNA isolated from such lysates demonstrates only minimal binding to oligothymidylic-cellulose and does not serve as a template for reverse transcription or in vitro translation. However, the extraction of frozen granulocytes into phenol in the presence of both heparin and vanadyl ribonucleoside allows the purification of relatively large quantities of RNA, which serves as an excellent template for reverse transcription and in vitro translation. Purification of granulocyte RNA by this method will facilitate study of granulocyte gene expression.

Myeloid-associated antigen 3-alpha-fucosyl-N-acetyllactosamine (FAL): location on various granulocyte membrane glycoproteins and masking upon monocytic differentiation.

Seven different granulocyte-reactive murine monoclonal antibodies (mAb) were studied. The antigens recognized by these mAb were immunoprecipitated from lysates of 125I-labeled granulocytes of healthy donors. The isolated antigens were analyzed by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel and autoradiography. All 7 antibodies precipitated the same 6 membrane polypeptides from membrane-iodinated granulocyte lysates: 105 and 150-kDa as most pronounced, together with 260-, 230-, 67- and 52-kDa polypeptides. One of the antibodies studied, B4.3, is directed against 3-alpha-fucosyl-N-acetyllactosamine as shown by absorption with the synthesized carbohydrate molecule. Competition experiments with 125I-labeled B4.3 demonstrated complete inhibition of binding by B4.3 and 3 of the other antibodies (VM D5, UJ308, MI/N1) and partial inhibition by the 3 other antibodies (FMC 10, FMC 12, FMC 13), indicating binding to the same antigenic structure. None of the 7 mAb reacted with monocytes in the immunofluorescence technique, but after neuraminidase treatment of these cells, positive reactions were obtained with all mAb. Immunoprecipitation with lysates of both native and neuraminidase-treated monocytes showed no polypeptide bands. Monocytic differentiation of the cell line HL60 by 12-O-tetradecanoylphorbol-13-acetate (TPA) and of cell line U 937 by dimethylsulfoxide and TPA was accompanied by a decrease in reactivity with these antibodies, which could be recovered by neuraminidase treatment. This indicates that 3-alpha-fucosyl-N-acetyllactosamine is masked for the detection of the antibody upon monocytic differentiation by sialylation.

The analysis of cytostatic activity of human peripheral blood granulocytes and its augmentation with Nocardia rubra cell wall skeleton (N-CWS).

Granulocytes of normal volunteers showed substantial levels of cytostatic activity against tumor-cell-derived cell lines. Spontaneous cytostatic activity of the granulocytes was observed after 24 h incubation and reached the maximal level within 48 h. Lysates of granulocytes also showed significant cytostatic activity. The cytostatic activity of the granulocytes was significantly augmented by in vitro incubation with Nocardia rubra cell-wall skeleton (N-CWS). Maximal augmentation of granulocyte-mediated cytostasis was achieved with 2.5 micrograms N-CWS per ml after 24 h incubation when the granulocyte-to-tumor-cell ratio was 10:1. The N-CWS itself showed no direct effect on the uptake of 3H-thymidine by tumor cells.

A study of the effect of some drugs which cause agranulocytosis on the biosynthesis of pyrimidines in human granulocytes

The activities of the first five enzymes of de novo pyrimidine biosynthesis [carbamoylphosphate: l-aspartate transferase (EC 2.1.3.2), l-4,5-dihydroorotate aminohydrolase (EC 3.5.2.3), l-4, 5-dihydroorotate, O 2 oxidoreductase (EC 1.3.3.1), orotidine-5'-phosphate-pyrophosphate phosphoribosyltransferase (EC 2.4.2.10) and orotidine-5'-phosphate carboxy-lyase (EC 4.1.1.23)] have been measured in human granulocytes. All five enzymes can be measured in the granulocytes (approximately 5 × 10 7 cells) from 20 ml blood using the ultrasensitive radiochemical methods described. The effect of amidopyrine, carbimazole, chloramphenicol, chlorpromazine, phenylbutazone and methylthiouracil on the activity of these enzymes in human granulocyte lysates has been investigated. Phenylbutazone inhibited l-4,5-dihydroorotate, O 2 oxidoreductase (EC 1.3.3.1). Carbimazole either inhibited or stimulated carbamoylphosphate, l-aspartate transferase (EC 2.1.3.2) depending on the experimental conditions. Thus, when the substrate and inhibitor concentrations were held constant and the amount of the enzyme in the system varied, the drug was stimulatory at very low enzyme concentrations and inhibitory at higher enzyme concentrations. No other examples of enzyme inhibition or activation were encountered.