Normal Human Blood Cells Research Articles

CDNA Cloning and Characterization of Human Monocyte/Macrophage Serine Esterase-1

Human monocyte/macrophage serine esterase (HMSE), commonly known as acid esterase or alpha-naphthylacetate esterase, comprises a group of five enzyme variants that can be distinguished by their isoelectric points from esterase variants of the other normal human blood cell populations. A cDNA for one of the monocytic enzyme variants (HMSE1) was cloned from a U-937 lambda gt11 cDNA library by screening with an oligonucleotide mixture designed according to amino acid sequence data of the purified enzyme. The cDNA contains 1,727 bp with an open reading frame of 1,512 bp coding for a protein of 503 amino acid residues. HMSE1 cDNA represents the first cloned monocyte/macrophage-specific serine esterase and its sequence shows up to 77% homology to other known serine esterases of different species. The amino acid composition of the putative active site of HMSE1 as deduced from the nucleotide sequence corresponds with the active sites of other serine esterases but not with the active sites of serine proteases. Hybridization of the cDNA with RNA of separated normal blood cell populations and hematopoietic cell lines shows restricted expression within the monocyte/macrophage lineage.

Increased Methylation of the <i>c-fms</i>Protooncogene in Acute Myelomonocytic Leukemias

DNA methylation provides an epigenetic information possibly involved in differentiation processes and gene regulation. In this study we investigated the methylation state of the c-fms/M-CSF receptor gene in normal human blood cells and leukemias. The methylation pattern of the c-fms gene as detected by isoschizomeric restriction analysis with MspI/HpaII showed only slight interindividual variations in normal donors, but there were constant differences between granulocytes and monocytes from the same donor. Of 21 acute myelomonocytic leukemias investigated, 85% revealed a methylation pattern different from that of normal monocytes. One or more restriction sites which were at least partly unmethylated in normal monocytes proved to be methylated in leukemic cells. In contrast, leukemias of lymphocytic origin showed hypomethylation of the c-fms gene. There was no correlation between the methylation state of the c-fms gene and its expression at the RNA level, but an increase in monocyte/macrophage-specific differentiation markers could be observed in those samples which exhibited a methylation pattern similar to that of normal monocytes. The increased DNA methylation within the c-fms gene might reflect the inactivation of differentiation genes in leukemic cell clones, contributing to their maturation arrest. Furthermore, neoplastic hematopoietic cells seem to exhibit lineage-specific differences in c-fms gene methylation.

Gamma-ray- and Fission Neutron-induced Micronuclei PHA Stimulated and Unstimulated Human Lymphocytes.

Two groups of normal human blood cells, one stimulated with phytohaemagglutinin (PHA) for 24 hr (G1-S phase of the cell cycle) and one unstimulated (G0 phase), were irradiated with 60Co gamma rays or 252Cf radiation. A comparison of radiation-induced micronucleus frequencies showed that the high-dose-rate gamma rays were more effective in inducing micronuclei than low-dose-rate gamma rays. In the cells exposed to low-dose-rate irradiation, there was little difference between the frequency of micronuclei in the G0 phase and the G1-S phase. However, cells in the G1-S phase were more sensitive than G0-phase cells to high-dose-rate gamma rays. The relative biological effectiveness of 252Cf neutron irradiation measured in micronucleus assays was consistent with the value obtained for the lethal effect of 252Cf on cultured cells.

Negative regulator of pluripotent hematopoietic stem cell proliferation in human white blood cells and plasma as analysed by enzyme immunoassay

This paper describes the analysis, by a highly sensitive and specific enzyme immunoassay (EIA), of AcSDKP, a tetrapeptide recently isolated from fetal calf bone marrow and subsequently purified and identified which substantially inhibits entry into cycle of hematopoietic pluripotent stem cells (CFU-S). This molecule has a marked protective effect in mice during anticancer chemotherapy with phase-specific drugs and plays an essential role in maintaining CFU-S out of cycle in normal mice. Using acetylcholinesterase-AcSDKP conjugate as tracer, rabbit specific antiserum and 96-well microtiter plates coated with a mouse monoclonal anti-rabbit IgG antibody, this EIA allows detection of AcSDKP at 15 fmol levels with a coefficient of variation less than 10% in the 50–500 fmol range. When combined with high-performance liquid chromatography, this assay clearly reveals the presence of this peptide in normal human white blood cells whereas in supernatant from cultured lymphocytes and in plasma the immunoreactive material is distinct from standard AcSDKP.

Tyrosine protein kinase activity in normal and leukaemic human blood cells.

Tyrosine protein kinase (TPK) activity was measured in subcellular fractions of normal granulocytes, lymphocytes and monocytes, and acute and chronic myeloid and lymphoid leukaemic cells. Of several tested tyrosine-containing substrates, poly (glutamic acid: tyrosine = 4:1) (S1) proved to be the best synthetic substrate. High cytosolic TPK activity was found in every cell type. Different TPKs may exist in various cell fractions, as was indicated by the difference in Km values for S1 in cell fractions of normal granulocytes and lymphocytes. No significant difference was found in total TPK activity between normal and leukaemic cells, indicating that total TPK activity is not related to the leukaemic process itself. A highly significant difference was found in membrane fractions in normal granulocytes and M1-M2 AML cells versus normal monocytes and M4-M5 AML cells, suggesting an association between TPK activity and monocytic differentiation in these cell fractions.

A monoclonal antibody 1A6 to T‐cell recognizes a common antigen expressed on lymphocytes from normal and leukemic patients, and inhibits nk cell activity

A new monoclonal antibody (MAB), IA61gG2a, has been derived from Balb/c mice after immunization with T-cell chronic lymphocytic leukemia (T-CLL). This monoclonal antibody, 1A6 was tested on normal human peripheral blood cells, leukemia patients' cells and leukemia-lymphoma cell lines. The results demonstrated that this antibody reacts with normal human T and B cells, but not with erythrocytes, granulocytes or platelets. Further examinations showed 10/11 leukemic patient cell types were positive with 1A6. In contrast, many leukemia-lymphoma cell lines were negative (14/17), i.e. T and B leukemia lymphoma cell lines, non-T and non-B leukemia cell lines as examined by complement mediated cytotoxicity test and immunofluorescence test. Other prominent characteristics of 1A6 MAB have been demonstrated, which include inhibition of myeloid colony formation (CFU-C) and suppression of human natural killer cell cytotoxicity (NKCC). These results suggest that 1A6 is a common marker for normal T, B, NK cells and leukemic patients cells having T and B cell origin, but 1A6 antigen is not expressed on many leukemia lymphoma cell lines. Therefore, it could be useful for studying malignant transformation of lymphoid cells.

Heat shock proteins in normal and leukemic blood cells.

Heat shock proteins (HSPs) are though to represent a ubiquitous cellular response to heat or stress. We tested whether HSPs can be induced in hairy cells, other human leukemic cells, and normal lymphocytes, and whether there are additive or synergistic effects between heat shock and interferon-alpha 2b (IFN-alpha 2b) on these cells. We analyzed lysates of cells from 22 patients (6 with hairy cell leukemia, 12 with other acute and chronic leukemias and lymphocytes of 4 normals) after exposure to a heat shock and/or IFN-alpha 2b by one-dimensional polyacrylamide gel electrophoresis. In all cells a pattern of HSPs was readily induced with prominent bands identified at approximately 115, 90, and 65 kD. None of these major proteins appeared to represent the previously described IFN-alpha 2b induced band at 80 kD. IFN-alpha 2b by itself was not found to induce HSPs. We conclude that a pattern of HSPs can readily be induced in a variety of normal and leukemic human blood cells. IFN-alpha 2b is not a HSP inducer in these cells. The previously described IFN-induced p80 is apparently not a HSP.

Human immunodeficiency virus infection studied in CD4-expressing human-murine T-cell hybrids

Human immunodeficiency virus (HIV) infection was studied by means of CD4-expressing human-murine T-cell hybrids, containing a variable amount of human chromosomes. Fusion of the HPRT − murine cell line BW5147 with human T-cell acute lymphoblastic leukemia or normal human blood cells resulted in a panel of human-murine T-cell hybrids. For this study, we used four hybrids containing all or several human chromosomes, which all expressed the CD4 antigen, as assessed by different anti-CD4 monoclonal antibodies (e.g., OKT4A, Leu-3a, and MT151) and, in addition, a variable number of other human T-cell antigens. For infection, HTLV-IIIB-infected H9 cells, pretreated with mitomycin C, and cell-free concentrated supernatants from these cells were used. In cells, of inoculated cultures of the CD4+ T-cell hybrids, no viral antigen could be demonstrated. Culture supernatants of inoculated hybrids, except for an initial rise due to the virus inoculum, never showed reverse transcriptase activity above background. Cocultivation of these cell cultures with H9 cells did not result in detectable virus replication. Cocultivation of CD4-expressing hybrid cells with HIV-infected cells did not result in syncytium formation. Moreover, these hybrids were resistant to infection with vesicular stomatitis virus (VSV)-HIV pseudotypes. These findings imply that expression of the CD4 antigen on the cell surface is not sufficient for productive infection with HIV. The infectivity block observed in these hybrids seems to occur at the level of virus penetration, presumably at the stage of membrane fusion events.

A BglII RFLP demonstrated for the II-3 gene in normal human blood cells and leukemias

A BglII RFLP demonstrated for the II-3 gene in normal human blood cells and leukemias

Isotype-specific suppression of IgE synthesis in a human myeloma cell line (U-266) by an IgE-binding protein derived from normal human peripheral blood cells.

Human peripheral blood cells produce immunoglobulin-binding factors upon stimulation with purified protein derivative. The binding factors from 2 healthy individuals were purified by IgE affinity chromatography. IgE-suppressive activity was demonstrable using the IgE-producing human myeloma cell line U-266 as an assay system. The effect of the suppressive factor seems to be reversible, isotype specific, as the immunoglobulin synthesis in the IgGk-producing U-1958 myeloma line was not affected, and selective for the epsilon heavy chain. The results also suggest that this suppression is not the result of a decrease in DNA synthesis or suppression of protein synthesis in general.

Characterization of a human follicular thyroid carcinoma cell line (UCLA RO 82 W-1)

A thyroid tumor cell line has been established from the metastases of a follicular carcinoma in a female patient. Although the primary tumor released thyroglobulin (Tg) into the circulation (greater than 10,000 ng/ml), the uptake of I131 was less than 2%. After 37 replications the doubling time was 4 days and confluency was reached after 7 days from inoculation of 3 x 10(7) cells. This human thyroid tumor cell line has now been growing in culture for several years. An aneuploid chromosomal pattern was observed (62-82 chromosomes). A pair of X chromosomes was present but no Y chromosome was found which is compatible with the female origin of the cell line. EM studies revealed the presence of microvilli. Immunoperoxidase staining using specific anti-human Tg antisera indicated the presence of Tg within the cells. Nude mice developed solid-cystic tumors within 6 months after injection of the cells. The basal release of immunodetectable Tg, as measured in a perifusion system, increased in response to thyroid stimulating hormone (TSH) (P less than 0.025) or TSH combined with theophylline (P less than 0.001). Unusual isoenzyme patterns for galactose-1-phosphate-uridyltransferase (GALT) and phosphoglucomutase1 (PGM1) were detected in the tumor, compared with normal human fibroblasts and blood cells and isoenzyme patterns from the patient's lymphocytes. Because this malignant human thyroid follicular cell line has retained the ability to synthesize Tg it represents a valuable model for the study of human follicular carcinomas.

A murine monoclonal antibody raised against human non-small cell carcinoma of the lung.

Murine monoclonal antibodies (MAbs) reactive with human non-small cell carcinoma of the lung (NSCCL) were produced following immunization with a membrane preparation of Calu-3, a human adenocarcinoma of the lung cell line grown in nude mice. Positive hybrids unreactive with normal liver membranes and human peripheral white blood cells were selected for further testing. One MAb (RS5-4H6) recognized an antigen expressed in a variety of lung and other carcinoma cell lines as detected by flow cytometry. Immunohistology showed a selectivity for normal and neoplastic lung epithelium, as well as other cancers. The antigen was detected by immunohistology in 87% of tumor specimens derived from the lung, breast, colon, kidney, and ovary. Most other normal human tissues were not stained but occasional cells of the stomach, salivary and other glands, as well as kidney tubules were reactive. This MAb is an IgG1. Western blot analysis indicated that the antibody reacts selectively with an antigen greater than 300 kD. The antigen is resistant to neuraminidase treatment and periodate oxidation, but sensitive to pronase treatment, suggesting that the epitope is peptide in nature. This antibody may be potentially useful as a targeting agent for radioimmunodetection and immunoconjugate therapy.

The Protooncogene c-fos Is Transcriptionally Active in Normal Human Granulocytes

AbstractTotal cellular RNA of highly purified normal human blood cell populations was analyzed for the expression of the protooncogene c-fos, the cellular counterpart of the transforming FBJ virus. In marked contrast to previous findings based on in vitro studies with permanent leukemic cell lines, c-fos transcription was restricted to granulocytes. Neither blood monocytes nor blood lymphocytes or alveo- lar macrophages revealed detectable levels of c-fos transcription. Whereas this cellular oncogene is constitutively expressed in granulocytes, the monocytic cell line U-937 showed a transient c-fos transcription only after induction of differentiation. The contradiction between the results found in vivo and in vitro is discussed.

The protooncogene c-fos is transcriptionally active in normal human granulocytes

Total cellular RNA of highly purified normal human blood cell populations was analyzed for the expression of the protooncogene c-fos, the cellular counterpart of the transforming FBJ virus. In marked contrast to previous findings based on in vitro studies with permanent leukemic cell lines, c-fos transcription was restricted to granulocytes. Neither blood monocytes nor blood lymphocytes or alveolar macrophages revealed detectable levels of c-fos transcription. Whereas this cellular oncogene is constitutively expressed in granulocytes, the monocytic cell line U-937 showed a transient c-fos transcription only after induction of differentiation. The contradiction between the results found in vivo and in vitro is discussed.

Calmodulin in lymphocyte mitogenic stimulation and in lymphoid cell line growth

Calmodulin levels are elevated three- to fourfold in the dividing cells, resulting from the lectin-induced stimulation of fresh human lymphocytes. This increase in calmodulin appears to be related mainly to progression into S phase and supports the hypothesis that calmodulin might be crucial in regulating the progression of lymphoblasts through their division cycle. Calmodulin levels are higher in a lymphoid cell line derived from human acute lymphoblastic leukemia blood cells than in a lymphoid cell line derived from normal human blood cells, suggesting that calmodulin could be an important mediator of the leukemogenetic process.

Tyrosine kinases in normal human blood cells: Platelet but not erythrocyte band 3 tyrosine kinase is p60 c-src

The major tyrosine kinase from platelets was purified as a 51 kDa active enzyme which was shown to be a degradation product of the protooncogene product p60 c-src. Immuno-depletion experiments using a monoclonal antibody recognizing p60 c-src failed to remove band 3 phosphorylating activity from red blood cell membranes. The erythrocyte tyrosine kinase was not at all immunoprecipitated by this antibody under conditions where the platelet enzyme was immunoprecipitated.

Analysis of nuclear m-AMSA content by DNA fluorochrome competition

Spectrofluorometry and flow cytometry were used to measure cellular uptake of the anti-leukemic drug 4′-(9-acridinylamino) methane sulfon-m-anisidide (m-AMSA). Because of its very low intrinsic fluorescence, we used m-AMSA to quench DNA-fluorescence induced by the vital DNA fluorochrome Hoechst 33342. Maximum fluorescence was obtained with cells incubated in the fluorochrome alone. Subsequent incubation of cells in increasing concentrations of m-AMSA resulted in a gradual decrease in fluorescence. Upon incubation in drug-free medium, the quenching phenomenon was reversible, consistent with rapid exit of m-AMSA from the cells. The novel competitive fluorescence assay for cellular uptake for m-AMSA showed a better correlation to nuclear accumulation of the drug, than to its overall cellular accumulation, which may be important in assessment of cellular resistance to m-AMSA, with possible low nuclear accumulation of the drug. When this competitive fluorescence technique for measurement of cellular m-AMSA concentration was applied in flow-cytometric setting, subpopulations of normal human white blood cells were detected with distinctly different fluorescence patterns, indicating differences in cellular m-AMSA uptake. The potential use of this technique is to detect differences between cell subpopulations with different drug uptake abilities.

Release of granulocyte-macrophage colony-inhibiting activity by normal human postthymic precursor cells.

Seven normal human peripheral blood cell fractions (buffy coat, mononuclear cells, non-T, T, Fc-IgM receptor-depleted T-lymphocyte, Fc-IgG receptor-depleted T-lymphocyte, and autologous rosette-forming T-cell-depleted T-lymphocyte subpopulations) treated with phytohemagglutinin (PHA) were examined for the production of granulocyte-macrophage colony-stimulating activity (CSA). It was found that medium conditioned by a T-lymphocyte subpopulation depleted of autologous rosette-forming T-cells (Tar cells, a postthymic precursor subpopulation that inhibits Ig synthesis) stimulated colony-forming units of granulocyte and macrophages (CFU-GM) to a greater extent than did the other conditioned media (CM) analyzed. Based on this finding, CM from an enriched Tar subpopulation was prepared and thus showed that PHA-treated Tar cells release a factor capable of inhibiting CFU-GM growth. The inhibitory activity of this factor persisted-after heat inactivation, suggesting that cause of the colony-inhibiting activity (CIA) is other than interferon. Further studies revealed that Tar-derived inhibitory factor acts either directly upon CFU-GM or via monocytes/macrophages (M phi/Ma), enhancing CIA, and not the level of CSA production by M phi/Ma. The overall data are interpreted as demonstrating the presence of CIA in a specific T-lymphocyte subpopulation that may represent a new relationship between lymphocytic and myelocytic systems in the human.

Nucleotide sequence analysis of a variant human T-cell leukemia virus (HTLV-Ib) provirus with a deletion in pX-I.

A variant of human T-cell leukemia virus subgroup I (HTLV-I), designated HTLV-Ib, has been isolated from a transformed T-lymphocytic cell line established from a Zairian patient with adult T-cell lymphoma. A recombinant phage clone of the variant provirus, denoted lambda MC-1, hybridizes under high stringency to HTLV-I DNA probes, but 17 of 43 restriction enzyme sites differ from those of HTLV-I, 10 of them clustering within 1.5 kilobases in the env-pX region. Since this variant virus retains its capacity to transform T-cells in vitro, and since a pX product is suspected to be important in transformation, we have determined the nucleotide sequence of the entire pX region of this virus for comparison to the prototype HTLV-I. In addition, the region between the gag and pol genes, parts of the pol and env genes, and a portion of the U3 region of the long terminal repeat sequence were also analyzed. We noted 141 single-base-pair changes among 3,897 base pairs, which were relatively well distributed over those portions of the provirus that were examined. In addition, an 11-base-pair deletion was found which included the potential initiator ATG codon of the first open reading frame of pX (pX-I). The next potential initiator codon predicted by the sequence is followed by 10 codons and then a termination codon. An identical deletion was also demonstrated in the only provirus present in another cell line established from the same patient on a different occasion after transformation in vitro of normal human umbilical cord blood cells. These results indicate that pX-I is not required for transformation.

Nucleotide profiles of normal human blood cells determined by high-performance liquid chromatography

An anion-exchange high-performance liquid chromatography method has been used to quantitate the intracellular purine and pyrimidine nucleotides in extracts of pure lymphocytes, monocytes, neutrophils, eosinophils, erythrocytes, and platelets isolated from the blood of healthy human donors. For accurate and reproducible measurements of the nucleotide profiles in different types of pure leukocytes, the cell suspensions have to be free of platelets and erythrocytes. Incubation of the purified leukocytes for 1 h at 0°C did not alter the nucleotide concentrations but reduced the interdonor variation to 10%. Incubation of purified lymphocytes for 1 h at 37°C caused considerable changes in the relative concentrations of the adenine, guanine, uracil, and cytosine nucleotides. During this incubation the cell viability, the cell number, and the ATP:ADP ratio decreased. Incubation of monocytes and granulocytes for 1 h at 37°C caused considerable loss of cells and/or cell death. For erythrocytes and platelets reproducible nucleotide concentrations were obtained after extraction of freshly isolated cells. During storage of erythrocytes, both at 0°C and at 37°C, a decrease in the ATP:ADP ratio was detected. In all cell types the predominant nucleotides were purine nucleotides, especially adenosine triphosphate. The relative concentrations of the adenine, guanine, uracil, and cytosine nucleotides were very reproducible per cell type and appeared to be characteristic for each cell type. The total nucleotide content was nearly the same for all cell types except erythrocytes, when expressed per microgram of protein. The described methods for purification and storage of blood cells will be useful for comparison of blood cells from healthy donors with those of patients, for example, leukemia patients, in which deviations of the purine and pyrimidine metabolic enzymes have already been described.