GATA and NF-Y participate in transcriptional regulation of FcgammaRIIA in megakaryocytic cells.

Human FcgammaRIIA, expressed on platelets, neutrophils, and macrophages, plays a major role in platelet activation and immune clearance. Clinical observations indicate that regulation of expression of this receptor is an important factor influencing the course of immune thrombocytopenia. We used both transient transfection with FcgammaRIIA promoter constructs and electrophoretic mobility shift assays (EMSA) to study the regulation of FcgammaRIIA transcription. In HEL (erythromegakaryocytic) cells, the 200 bp immediately 5' of the ATG start codon accounted for the majority of the activity of a 3.6-kb promoter fragment. Putative GATA (-161) and NF-Y (-119) sites are present. EMSA analyses demonstrate specific binding of both GATA-1 and GATA-2 to labeled oligonucleotides containing the putative GATA site with HEL but not U937 (myelomonocytic) nuclear extracts. Antibodies to NF-Y supershift the specific -119 NF-Y complex with HEL, U937, Jurkat (T-lymphocytic), and HeLa (nonhematopoietic) nuclear extracts. Comparison of the activity of GATA and NF-Y mutant constructs in HEL and U937 demonstrates that while either GATA or NF-Y mutation results in a large decrease in the promoter activity (2.2- and 2.3-fold, respectively) in HEL cells, neither mutation is effective in reducing activity in U937 cells. This is the first example of a promoter active in the megakaryocyte lineage in which NF-Y cooperates additively with GATA factors to regulate transcription. Identification of other factors that must be operational for FcgammaRIIA transcription in myelomonocytic cells which lack GATA factors will bolster our ongoing efforts to dissect the function of these Fc receptors in megakaryocytic and myelomonocytic cells in vivo.

The role of the human Fc receptor Fc gamma RIIA in the immune clearance of platelets: a transgenic mouse model.

In humans, the Fc receptor for IgG, FcgammaRIIA, is expressed on macrophages and platelets and may play an important role in the pathophysiology of immune-mediated thrombocytopenia. Mice lack the genetic equivalent of human FcgammaRIIA. To better understand the role of FcgammaRIIA in vivo, FcgammaRIIA transgenic mice were generated and characterized. One transgenic mouse line expressed FcgammaRIIA on platelets and macrophages at levels equivalent to human cells, and cross-linking FcgammaRIIA on these platelets induced platelet aggregation. Immune-mediated thrombocytopenia in this transgenic line was studied using i.v. and i.p. administration of anti-mouse platelet Ab. In comparison with matched wild-type littermates that are negative for the FcgammaRIIA transgene, Ab-mediated thrombocytopenia was significantly more severe in the FcgammaRIIA transgenic mice. In contrast, FcR gamma-chain knockout mice that lack functional expression of the Fc receptors FcgammaRI and FcgammaRIII on splenic macrophages did not demonstrate Ab-mediated thrombocytopenia. We generated FcgammaRIIA transgenic x FcR gamma-chain knockout mice to examine the role of FcgammaRIIA in immune clearance in the absence of functional FcgammaRI and FcgammaRIII. In FcgammaRIIA transgenic x FcR gamma-chain knockout mice, severe immune thrombocytopenia mediated by FcgammaRIIA was observed. These results demonstrate that FcgammaRIIA does not require the FcR gamma-chain for expression or function in vivo. Furthermore, taken together, the data suggest that the human Fc receptor FcgammaRIIA plays a significant role in the immune clearance of platelets in vivo.

Lineage-specific alternative splicing of the human Fc gamma RIIA transmembrane exon requires sequences near the 3' splice site.

The human Fc gamma RIIA gene produces multiple transcripts, including those with (Fc gamma RIIa1) and without (Fc gamma RIIa2) the single exon encoding the transmembrane domain (TM). Previously, a fluorescence-based RT-PCR assay showed lineage-specific differences in Fc gamma RIIA transcript ratios (Fc gamma RIIa2/Fc gamma RIIa1). The mechanism of this lineage-specific expression was investigated in this study. Differential transcript stability does not play a major role, because transcript ratios remained constant in cells with both low (K562) and high (Dami) ratios following actinomycin D treatment. Transient expression studies in K562 and Dami cells using a minigene construct containing a 5.0 kb genomic fragment including the TM exon and adjacent intron and exon sequences showed recapitulation of endogenous transcript ratios. The TM exon was efficiently spliced in by the constitutive splicing machinery in HeLa cells, an Fc gamma RIIA-negative cell line. Lineage-specific TM exon skipping was markedly diminished by two independent minigene mutations: a point mutation of the first nucleotide of the TM exon, and a five basepair intronic deletion near a putative branchpoint. These data demonstrate that cis-acting sequences in or near the TM exon 3' splice acceptor site contribute to lineage-specific differences in Fc gamma RIIA transcript ratios.

Fluorescence-based RT PCR analysis: determination of the ratio of soluble to membrane-bound forms of Fc gamma RIIA transcripts in hematopoietic cell lines.

We have developed a fluorescence-based RT PCR assay for determination of the ratio of two alternatively spliced transcripts in different cell types. Fluorescence detection, by an automated DNA sequencer, allows enhanced sensitivity and ease of data processing. PCR products are fluorescently tagged using a dye-labeled oligonucleotide primer during the PCR reaction. Assay conditions were first defined so that fluorescence intensity of the PCR products was linear with respect to input RNA and exponential relative to PCR cycle number. Sensitivity and reproducibility of detection were evaluated with serial dilutions of RT PCR reactions. We have applied this assay to an analysis of the lineage-specific expression of two human Fc gamma RIIA transcripts, Fc gamma RIIa1 and Fc gamma RIIa2, in different hematopoietic cell lines. Previously, we noted that when standard RT PCR conditions are used with primers that bracket the TM exon, the pattern of expression of these transcripts as assessed by ethidium bromide staining of agarose gels varied in different hematopoietic cell lineages. Using the fluorescence-based RT PCR method, we now confirm our previous findings and quantitate transcript ratios (Fc gamma RIIa2/Fc gamma RIIa1) in several hematopoietic cell lines. The ratio varies from 0.70 (41% Fc gamma RIIa2) in the erythroleukemic cell line HEL, to 0.14 (12% Fc gamma RIIa2) in the monocytic cell line U937, to 0.07 (6% Fc gamma RIIa2) in the multipotential cell line K562. This fluorescent RT PCR method provides a general approach to quantitating mRNA levels and ratios of PCR products in other gene systems.

A soluble form of the human Fc receptor Fc gamma RIIA: cloning, transcript analysis and detection.

The Fc gamma receptors (Fc gamma R) are glycoproteins that bind the Fc region of immunoglobulin G. Human hematopoietic cells express three biochemically distinct classes of Fc gamma receptors: Fc gamma RI (CD64), Fc gamma RII (CD32) and Fc gamma RIII (CD16). Complementary DNA (cDNA) clones for each of the human Fc gamma receptors have been isolated from myeloid and lymphoid cells. We describe the isolation and characterization of four Fc gamma RII clones from a cDNA library obtained from a megakaryocyte-like cell line, human erythroleukemia (HEL). Three clones encode the Fc gamma RIIA transmembrane (TM) form, while one novel clone lacks the TM region but retains the cytoplasmic domain. By conducting reverse transcription coupled to polymerase chain reaction (PCR), we found transcripts coding for this unique form of receptor in RNA from platelets, HEL cells and a second megakaryocyte-like cell line, CHRF-288-11. These results were confirmed by RNase protection analysis of RNA from HEL cells. The structure of the novel cDNA suggested that it codes for a soluble form of Fc gamma RIIA. A soluble Fc gamma RII protein was detected in the conditioned medium from HEL cells but not from the Fc gamma RII-negative T cell line, Jurkat, by immunoprecipitation with the anti-Fc gamma RII monoclonal antibody (mAb), IV.3. The immunoprecipitated protein was of the expected size for a soluble Fc gamma RII lacking the TM region but retaining the cytoplasmic domain. Soluble Fc gamma RIIA may be important in modulating the interaction between immune complexes and membrane-associated Fc gamma RII.

Differential expression of Fc gamma RIIA, Fc gamma RIIB and Fc gamma RIIC in hematopoietic cells: analysis of transcripts.

Fc gamma receptors (Fc gamma R) are glycoproteins that function in the immune response through their ability to bind the Fc portion of immunoglobulin G. Of the three human Fc gamma R classes, Fc gamma RII is most widely distributed among hematopoietic cells and is the only Fc gamma R class present on platelets and megakaryocytes. There are three different genes coding for Fc gamma RII: Fc gamma RIIA, Fc gamma RIIB and Fc gamma RIIC. Alternative splicing of at least two of these genes results in the production of multiple transcripts. Combining Northern blot analysis with reverse transcription-PCR, we analyzed steady state levels of Fc gamma RII mRNA in the megakaryocytic, myeloid and lymphoid lineages. We determined that megakaryocytic cells predominantly contain Fc gamma RIIA mRNA; Fc gamma RIIA transcripts with and without the transmembrane exon (Fc gamma RIIa1 and Fc gamma RIIa2, respectively) are present in comparable amounts. In contrast, B lymphocytes do not express Fc gamma RIIA mRNAs, but do contain both Fc gamma RIIB transcripts, Fc gamma RIIb1 and Fc gamma RIIb2, as well as the Fc gamma RIIC transcript, Fc gamma RIIc. Myelomonocytic cells contain mRNAs from all three Fc gamma RII genes, predominantly the Fc gamma RIIa1 transcript, both Fc gamma RIIb1 and Fc gamma RIIb2 transcripts and Fc gamma RIIc. Lineage-specific expression of the Fc gamma RII genes implies both differential regulation of expression and differential function in diverse cells.

Characterization of the 5'-flanking transcriptional regulatory region of the human Fc gamma receptor gene, Fc gamma RIIA.

The human Fc gamma receptor gene Fc gamma RIIA is expressed in platelets, neutrophils, monocytes and macrophages. Understanding the regulation of expression of Fc gamma RIIA will enhance our knowledge of regulated gene expression and immune function in these cells. We cloned a 3.65 kb region of the 5' end of the Fc gamma RIIA gene and characterized 3.4 kb of previously unreported sequence of the 5'-flanking region. Primer extension studies and RNase protection analyses of mRNA from HEL, K562 and U937 cells revealed multiple transcription start sites. One transcription start site mapped to a 5'-untranslated (5'UT) exon approximately 1 kb 5' to the ATG translation initiation codon, while a second start site mapped near the ATG codon. Reverse transcription combined with PCR (RT-PCR) employing an oligonucleotide in the putative 5'UT exon and an antisense oligonucleotide in the translated region yielded products which confirm that transcription starts in this 5'UT exon 881 bp upstream of the ATG codon. Sequence analysis of the RT-PCR products showed two related RNA splice products which use alternative 3'-consensus AG splice acceptor sites. Fc gamma RIIA mRNA thus has three distinct potential 5'UT regions, two alternatively spliced forms from the start site in the 5'UT exon and the third from the start site near the ATG codon. Comparisons of the human Fc gamma RIIA 5'-flanking region with human Fc gamma RI and mouse Fc gamma RII beta genes as well as with other genes expressed in megakaryocytes, neutrophils and monocytes reveal structural similarities and shared promoter elements.

Transient modulation and internalization of T4 antigen induced by phorbol esters.

Phorbol esters are known to alter the expression of surface antigens and receptors on a variety of mammalian cell types. On T lymphoblastoid cell lines and peripheral blood T cells, phorbol esters have been shown to selectively reduce the expression of the T4 antigen. To more fully characterize this process, we have examined the metabolic requirements for this phorbol ester effect, and have evaluated the relationship between phorbol ester-induced T4 loss and the expression of receptors for phorbol-12,13-dibutyrate (PDB) on purified peripheral blood T4 cells. We observed that the loss of T4 on peripheral blood lymphocytes (PBL) occurred at PDB concentrations at which 10 to 15% of phorbol ester binding sites were occupied. The loss of T4 was inhibited at 4 degrees C, and by azide, methylamine, and sodium fluoride, but not by inhibitors of DNA synthesis. When cells were exposed to phorbol esters for greater than 2 days, the T4 antigen was again expressed on the cell surface despite the continued presence of phorbol esters. Cells which had recovered T4 were resistant to the effects of freshly added PDB on this antigen, and this resistance correlated with a 55% reduction in phorbol ester binding sites. Studies on fixed PBL T4 cells and MOLT-4 cells by immunofluorescence microscopy demonstrated that the decreased expression of T4 from the cell surface correlated with a bright clustering of T4 within the cytoplasm, indicating that PDB had induced an internalization of this antigen. These observations demonstrate that the binding of phorbol esters to specific receptors on lymphocytes initiates metabolically dependent events which result in the internalization of the T4 antigen. These findings may be relevant to mechanisms by which T4 functions as a signal-transducing molecule in vivo.

The conformation of apolipoprotein E isoforms in phospholipid complexes and their interaction with human Hep G2 cells.

The human hepatoma cell line, Hep G2, has been used to compare the metabolism by isolated liver cells of purified isoforms of human apolipoprotein E (apo E). Complexes of [125I]apo E-3/3, 2/2, 3/2 and 4/3 with dimyristoyl phosphatidylcholine (DMPC) were prepared by a detergent-dialysis method: discoidal, bilayer complexes with a stoichiometry of 125 +/- 15 mol DMPC/mol apo E resulted. The predominant phenotype apo E-3/3, and the phenotype apo E-2/2 characteristic of patients with Type III hyperlipoproteinemia, interact similarly with DMPC and adopt the same conformation with 60-70% alpha-helix, as monitored by circular dichroism spectroscopy. The uptake and degradation at 37 degrees C, and binding at 4 degrees C by Hep G2 cells, of [125I]apo E-3/3/DMPC and [125I]apo E-2/2/DMPC complexes were compared. Apo E-3/3 was degraded more rapidly than apo E-2/2 suggesting that the diminished catabolism of the latter phenotype by intact livers is due to lack of recognition by the hepatocytes. The observed degradation of apo E was 3-4 times greater than that which could be attributed to fluid phase endocytosis and low-affinity adsorptive endocytosis. The degradation of [125I]apo A-I by Hep G2 cells can be accounted for by the above endocytotic mechanisms. The distinction between apo E-3/3 and apo E-2/2 isoforms is attributed to the presence of a cell-surface receptor on Hep G2 cells which binds apo E-3/3 with a higher affinity than apo E-2/2.

Phorbol ester modulation of T-cell antigens in the Jurkat lymphoblastic leukemia cell line.

The effect of phorbol dibutyrate (PDB) on the cell surface antigens of the human T-cell acute lymphoblastic leukemia cell line, Jurkat, was studied with OKT monoclonal antibodies by indirect immunofluorescence assay. Cells were analyzed in an Ortho Spectrum III fluorescence-activated flow cytometer. The surface antigen profile of untreated Jurkat cells resembled that of thymocytes; high levels of T3, T4, T6, T8, T9, T10, and T11 antigens were detected. Although 89% of cells were positive for T11, the putative sheep erythrocyte receptor, only 12% were able to form erythrocyte (sheep) rosettes. Exposure of the cells to 1.0 microM PDB for up to 7 days resulted in a rapid loss in T4 expression and a slower decrease in T6 reactivity, while the percentage of cells positive for T3, T8, T10, and T11 remained high. T4 reappeared on the cell surface when PDB was removed by washing. T11 antigen density increased 70%, and this was accompanied by an increase in the percentage of erythrocyte-rosetting cells from 12 to 55%. These changes in cell surface antigens induced by PDB suggested differentiation to a more mature state (i.e., a precursor cytotoxic-suppressor T-lymphocyte, T3+T8+T10+T11+). However, the reversibility of the change in T4 expression indicated that T4 loss was not a manifestation of terminal differentiation but rather was consistent with a phorbol ester-induced modulation of the cell surface T4 antigen.

Metabolism of odd-numbered, normal fatty acids in Tetrahymena pyriformis W.

Tetrahymena pyriformis W cells were grown with short- and long-chain, odd and even normal fatty acid supplements. Tris acetate addition had no effect on the fatty acyl composition of the glycerophospholipids or sphingolipids, while Tris propionate supplementation led to a marked increase in odd normal fatty acids at the expense of even normal acids in both classes of complex lipids. This enhancement of odd normal acids permitted the identification of 17:1 delta 9(n), 17:2 delta 6,9(n), 17:2 delta 9,12(n), 17:3 delta 6,9,12(n), 19:1 delta 9(n), 19:2 delta 9,12(n) and 19:3 delta 6,9,12(n) by oxidation with periodate-permanganate and examination of the short-chain fragments. Supplementation with pentadecanoic acid (15:0(n)) led to an increase in the proportions of normal C15, C17 and C19 acids. The increase in C15 acids primarily reflected a rise in 15:0(n), whereas the rise in the levels of C17 and C19 acids was accounted for by an elevation of unsaturated acids. Growth with heptadecanoic acid (17:0(n)) resulted in substantial increases in unsaturated normal C17 and C19 fatty acids, while nonadecanoic acid (19:0(n)) addition led only to an increase in the proportion of unsaturated C19 acids. Retroconversion of these saturated, odd normal long chain fatty acid supplements was limited. Supplementation with arachidic acid (20:0(n)) resulted in only a marginal increase (1.4%) in normal C20 fatty acids of both the glycerophospholipids and the mild alkali labile neutral lipids and provided no evidence that desaturation occurred. The release of 14CO2 from [1-14C]arachidic acid when incubated with the ciliates indicated that this long-chain saturate is accumulated, activated and degraded. Normal C16-C19 saturated fatty acids are substrates for the delta 9 desaturase. The delta 11 isomers arise by chain elongation. Normal C16-C19 delta 9 monoenoic acids are substrates for the delta 12 desaturase. Normal C16-C18 delta 9 monoenes, normal C16-C19 delta 9,12 dienes and 18:1 delta 11(n) are desaturated at the C-6,7 position.