Dissection of the role of macrophages in triggering T lymphocytes for interleukin 2 production by monoclonal antibody OKT3

Unfractionated human peripheral blood mononuclear cells produce a small amount of interleukin 2 (IL 2) by stimulation with a monoclonal anti-T3 antibody (OKT3) in vitro. The IL 2 production could be greatly augmented by the addition of a phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). In the presence of TPA, the T cell enriched fraction deprived of macrophages did not produce IL 2, but the T cells pulse-incubated with OKT3 and reconstituted with macrophages efficiently produced IL 2 in subsequent culture in the presence of TPA as did T cells reconstituted with OKT3-pulse-incubated macrophages. The stimulating effect of OKT3 in the presence of macrophages was inhibited dose-dependently by the addition of immunoglobulins, particularly by mouse IgG2a which is the same isotype as that of the OKT3 antibody, showing that it inhibits by blocking the binding of OKT3 to Fc receptors on macrophages. The same extent of IL 2 production was induced in T cells when paraformaldehyde-fixed macrophages were substituted for intact macrophages. Remarkable IL 2 production was also induced by OKT3 when latex beads coated with rabbit anti-mouse IgG2a antibody and TPA were added to the culture. It was confirmed that the production induced by these stimulations was due to an increase of IL 2 mRNA. These results show that effective signals for IL 2 production are generated by efficient crosslinking of T3 molecules which results from multi-interaction of T3 molecules on the T cell membrane and anti-T3 antibody molecules on macrophage membrane or on the surface of the latex particle.     

Polyclonal Antibody Production in Murine Spleen Cells Induced by Staphylococcus

Polyclonal plaque-forming cell (PFC) responses in murine spleen cells induced by Staphylococcus aureus and S. epidermidis were studied. Injection of Balb/c mice with S. aureus strain 248βH resulted in the generation of anti-trinitrophenyl (TNP) and anti-sheep red blood cell PFC in their spleens. Cultures of Balb/c spleen cells in the presence of S. aureus 248βH, Cowan I, or a protein A-deficient mutant yielded many anti-TNP PFC. The larger the number of organisms that were added to the cultures, the better was the PFC response. Both living and killed organisms, were capable of inducing the response, but an excess of living 248βH organisms in the cultures abrogated the response. All of the organisms (12 strains of S. aureus and 11 strains of S. epidermidis) freshly isolated from patients had the ability to induce the polyclonal PFC response in cell cultures. These organisms stimulated cultured C3H/HeJ mouse spleen cells, which were unresponsive to bacterial lipopolysaccharide (LPS). Cultured cells from the spleens of athymic nu/nu mice also responded to these organisms, and the number of PFC in nu/nu cell cultures was always greater than that in nu/+ cells prepared from a haired litter mate. Moreover, the responses of nu/nu spleen cell cultures to which nylon wool column-filtered splenic nu/+ T cells were added were lower than expected. These findings suggest that the polyclonal PFC response to staphylococci is thymus independent, but that the magnitude of the response is regulated by mature T cells. Cultures of macrophage-depleted spleen cells responded to the organisms to an extent similar to that of the control. The 248βH organisms were less capable of stimulating spleen cells of 2-week-old mice (i.e., early maturing B cells) than LPS. However, spleen cells from adult (7-week-old) and aged (9-month-old) mice responded well to both the organisms and LPS. Previous sensitization with the organisms in vivo did not affect any polyclonal responses of spleen cells in vitro to either the organisms or LPS. The role of staphylococcal protein A in the polyclonal PFC response to staphylococci is discussed.     

Primary structures of two types of alpha-subunit of rat brain Na+,K+,-ATPase deduced from cDNA sequences

A rat brain cDNA library was screened by using as a probe a fragment of cDNA encoding the alpha-subunit of human Na+,K+-ATPase. Two different cDNA clones were obtained and analyzed. One of them was concluded to be a cDNA encoding the alpha-subunit of the weakly ouabain-sensitive rat kidney-type Na+,K+-ATPase. The deduced amino acid sequence consists of 1,018 amino acids. The alpha-subunit of the rat kidney-type Na+,K+-ATPase shows 97% homology in amino acid sequence with the alpha-subunit of human, sheep, or pig enzyme and 87% with that of Torpedo. Based on a comparison of the amino acid sequence at the extracellular domain of the alpha-subunit between weakly ouabain-sensitive rat kidney-type enzyme and the ouabain-sensitive human, sheep, pig, or Torpedo enzyme, it was proposed that only two significant amino acid replacements are unique to the rat kidney-type alpha-subunit. Another cDNA clone obtained showed 72% homology in nucleotide sequence with the former cDNA coding the alpha-subunit of the rat kidney-type Na+,K+-ATPase and the deduced amino acid sequence exhibited 85% homology with that of the alpha-subunit of rat kidney-type Na+,K+-ATPase.     

Nuclear DNA content as an indicator of chemosensitivity in small-cell carcinoma of the lung

The relationship between the nuclear DNA histogram pattern of tumor cells obtained by bronchoscopic brushing and the response to combination chemotherapy (Cytoxan + Adriamycin + Vincristine) was studied in 28 patients with small-cell carcinoma of the lung. Microspectrophotometric analysis of the tumor cells showed a near-diploid nuclear DNA pattern in 18 patients and a hyperdiploid pattern in 10 patients. Eight of the ten patients with the hyperdiploid pattern showed a good response (complete or partial response) to the chemotherapy. However, of the 18 patients with the near-diploid DNA pattern, only 2 displayed a good response; the remaining 16 patients exhibited no response. The hyperdiploid DNA pattern of tumor cells was thus associated with a better response to chemotherapy than was the near-diploid pattern. These results indicate that the nuclear DNA histogram pattern may be an indicator for predicting the degree of response to chemotherapy in small-cell carcinoma of the lung.     

Differences in lectin binding patterns of normal human endometrium between proliferative and secretory phases

Lectin binding patterns in normal human endometrium were examined by light and electron microscopy using seven different lectins (ConA, WGA, RCA, PNA, UEA-1, DBA, and SBA). For light microscopic observations, criteria based on the incidence and intensity of cells positive for the lectin staining were adopted to evaluate the different staining patterns of the proliferative and secretory endometria obtained by the avidin-biotin-peroxidase complex (ABC) technique. At the light microscopic level, ConA, WGA, and RCA stained endometrial glandular cells in both phases. The number of PNA-positive cells with the binding sites entirely limited to the apical surface tended to be reduced slightly in the secretory phase. UEA-1 weakly stained the apical surface of glandular cells in the proliferative phase but not in the secretory phase. Among the lectins used in this study, DBA and SBA displayed remarkable changes between the phases. That is, in the proliferative phase they produced only a faint or slight positive stain at the apical surface, but the incidence and intensity of DBA- and the SBA-positive glandular cells increased in the secretory phase. By electron microscopy, the reaction product of ConA was observed in the plasma membrane, endoplasmic reticulum, nuclear envelope, and the Golgi apparatus, and the binding sites of RCA and DBA were observed in the plasma and Golgi membranes. Between both phases, the reactivity of ConA and RCA showed almost no change. However, the secretory endometrial cells containing the DBA-positive Golgi apparatus were markedly increased in number compared with the proliferative ones bearing the lectin-positive organelles.(ABSTRACT TRUNCATED AT 250 WORDS)     

A newly established human acute lymphoblastic leukemia cell line with characteristics of the earliest B-cell maturation

A new human acute lymphoblastic leukemia (ALL) cell line, designated HBL-3, was established from the bone marrow of a patient with non-T-ALL. The HBL-3 cell line expressed B4 (CD 19), BA-1 (CD 24) and HLA-DR antigens, but not surface immunoglobulin (SIg) or cytoplasmic immunoglobulin (CIg). The cell line lacked the common acute lymphoblastic leukemia antigen (CALLA) and antigenic markers characteristic of T-cell and myeloid cell lineages. The HBL-3 cells had structural rearrangements of both the homologous chromosome 9s, including a translocation with chromosome 1 which has been reported in a patient with common ALL. The cell line had rearranged immunoglobulin heavy chain genes but retained germ-line κ light chain genes and germ-line T-cell receptorβ- and γ-chain genes. The HBL-3 cell line was strongly positive for terminal deoxynucleotidyl transferase (TdT). These findings indicate that the HBL-3 cell line is derived from the earliest B-cell committed to B-cell lineage.     

Change in Gene Expression in Radish Cotyledons during Dark-Induced Senescence

Cotyledons detached from light-grown radish (Raphanus sativusL. cv. Comet) seedlings were used as a model system to studythe changes in nuclear gene expression during dark-induced senescenceof green leaves. Polyadenylated RNA was prepared from the cotyledonsat different times and then translated in a wheat germ system.Approximately 1,000 different polypeptides of the translationproducts were separated from each other by two-dimensional gelelectrophoresis. As judged from the density of autoradiographicspots of the translation products, the induction of senescenceby dark treatment involved an increase in 26 species, a decreasein 11 species, and a temporary increase and subsequent decreasein 8 species of translatable mRNA. A similar pattern of changein protein synthesis was also observed in the dark-treated cotyledonswhen the cotyledons were pulse-labeled with ³⁵S-methionine andthe soluble proteins separated by two-dimensional gel electrophoresis,though the polypeptide pattern on the gel did not coincide exactlywith those of the cell-free translation products. These findingsstrongly suggest that the process of leaf senescence is notsimply a passive and gradual death of the tissue, but involvesa drastic and sequential response of the cells to environmentalstimuli with respect to the gene expression of the cells. (Received July 21, 1987; Accepted September 30, 1987)     

Senescence-specific increase in cytosolic glutamine synthetase and its mRNA in radish cotyledons

Changes in the levels of cytosolic and chloroplastic isoforms of glutamine synthetase were examined in senescing radish (Raphanus sativus L. cv Comet) cotyledons by immunoblotting analysis using antibodies raised separately against maize glutamine synthetase isoforms. Translatable mRNAs for these isoforms were also examined by analyzing translation products from poly(A)⁺ RNA in a wheat germ system with the antibodies. The relative content of cytosolic isoform (GS₁) increased twofold in the cotyledons that were placed in the dark for 72 hours to accelerate senescence, while that of chloroplastic isoform (GS₂) declined to half of its initial level. The dark-treatment also increased the relative level of translatable mRNA for GS₁ sevenfold after 72 hours, and decreased rapidly that for GS₂ and for other nuclear-coded chloroplast proteins as well. Cotyledons also accumulated GS₁ mRNA when they became senescent after a lengthy growth period under continuous light. These observations suggested that GS₁ genes were activated, while those for GS₂ were repressed, and eventually the population of the enzyme was altered in senescent cotyledonary cells. The role of increased cytosolic enzyme is discussed in relation to the nitrogen metabolism in senescent leaves.     

Characterization of a glucuronyltransferase: neolactotetraosylceramide glucuronyltransferase from rat brain

The properties of a rat brain glucuronyltransferase, which is presumed to be associated with the biosynthesis of the HNK-1 epitope on sulfoglucuronyl glycolipids, are described. The enzyme required divalent cations for reaction, with maximal activity at 10mm Mn²⁺, and exhibited a dual optimum at pH 4–5 and pH 6 depending upon the buffer used, with the highest activity at pH 4.5 in MES buffer. This enzyme strictly recognized the Gal1-4GlcNAc terminal structure, and was highly specific for neolacto (type 2) glycolipids as acceptor. The enzyme was localized specifically in the brain, and was barely detected in other issues, including the thymus, spleen, liver, kidney, lung, and sciatic nerve fibres. Phosphatidylinositol and phosphatidylserine increased the enzymatic reaction 4.4- and 2.3-fold, respectively, whereas phosphatidylcholine slightly decreased the rate.Abbreviations GlcA glucuronic acid - Lc-PA₁₄ lactotetraose-phenyl-C₁₄H₂₉ - nLc-PA₁₄ neolactotetraose-phenyl-C₁₄H₂₉ - nLcOse₄-Cer neolactotetraosylceramide - NP-40 Nonidet P-40 - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - SGGL sulfoglucuronyl glycolipid     

Alterations in cardiac function and subcellular membrane activities after hypervitaminosis D3

The present study was designed to induce massive accumulation of calcium in the myocardium and to evaluate the effect of calcium overload on myocardial contractile function and biochemical activity of cardiac subcellular membranes. Rats were treated with an oral administration of 500,000 units/kg of vitamin D₃ for 3 consecutive days, and their hearts were sampled on the 5th day for biochemical analysis. On the 4th and 5th days, heart rate, mean aortic pressure, left ventricular systolic pressure and left ventricular dP/dt were significantly lowered in vitamin D₃-treated rats, demonstrating the existence of appreciable myocardial contractile dysfunction. Marked increases in the myocardial calcium (67-fold increase) and mitochondrial calcium contents (24-fold increase) were observed by hypervitaminosis D3. Mitochondrial oxidative phosphorylation and ATPase activity were significantly reduced by this treatment. A decline in sarcolemmal Na⁺, K⁺-ATPase activity was also observed, while relatively minor or insignificant changes in calcium uptake and ATPase activities of sarcoplasmic reticulum were detectable. Electron microscopic examination revealed calcium deposits in the mitochondria after vitamin D₃ treatment. The results suggest that hypervitaminosis D₃ produces massive accumulation of calcium in the myocardium, particularly in the cardiac mitochondrial membrane, which may induce an impairment in the mitochondrial function and eventually may lead to a failure in the cardiac contractile function.