Human mononuclear phagocyte-associated antigens: II. Lymphokine-inducible antigens on the macrophage cell line,U937

We have utilized the U937 macrophage cell line as a model system for analysis of human mononuclear phagocyte (MNP) differentiation. In addition to expressing membrane antigens shared with other MNP, U937 possesses an intrinsic ability to become “activated” upon exposure to lymphokines. A heteroantiserum produced against lymphokine-stimulated U937 (anti-U937L) was utilized to detect acquired or inducible membrane antigens expressed on “activated” U937. Absorption of this antiserum to remove antibodies to nonstimulated U937 (U937N) did not remove the reactivity of anti-U937L/U937N to lymphokine-stimulated U937 as determined by an ¹²⁵I-protein A radioimmunoassay. The lymphokine-inducible antigens were not detectable on resident, human peritoneal macrophages. In addition to expression of lymphokine-inducible antigens, treated U937 cells displayed alterations in both morphology and functional activity (antibody-dependent cellular cytotoxicity). Kinetic analysis of lymphokine-stimulated U937 indicated that antigen expression occurred as early as 1–2 hr after lymphokine exposure, plateauing at 16–18 hr of stimulation. The inducible antigens were susceptible to proteolytic degradation and expression was blocked by inhibitors of protein synthesis. Inducible antigens detectable by anti-U937L/U937N did not result from the expression of cryptic or buried membrane antigens. Thus, the U937 cell line can be utilized for production of antibodies useful in analysis of membrane antigen expression during differentiation within the MNP system.     

Human mononuclear phagocyte-associated antigens: I. Identification and characterization

Rabbit antisera were produced against a lymphokine-activated human macrophage cell line, U937 (αU937), and human peritoneal macrophages (αPEMØ). After absorption with AB erythrocytes, pooled platelets, and B-lymphoblastoid cell lines, both antisera reacted by microcytotoxicity, indirect immunofluorescence (IF), and radioimmunoassay (RIA) with adherence-purified human peripheral blood monocytes, splenic and peritoneal macrophages, and leukemic myelomonoblasts. A panel of normal human T lymphocytes, B lymphocytes, and erythroid-myeloid or lymphoblastoid cell lines failed to react with both αU937 and αPEMØ. Although both heteroantisera reacted against polymorphonuclear leukocytes (PMNs), after absorption with PMNs specific reactivity against mononuclear phagocytes remained. Absorption of αU937 and αPEMØ with myelomonoblastic leukemia cells (AMML) removed IF and RIA activity against both PMNs and monocytes but not against splenic and peritoneal macrophages. In contrast, absorptions of both heteroantisera preparations with splenic macrophages abolished their IF and RIA reactivity not only to splenic and peritoneal macrophages but also to peripheral blood monocytes and leukemic myelomonoblasts. These results are consistent with (1) both antisera defining specific monocyte/macrophage-associated antigens(s) which are distinct from MHC-coded HLA-A,B,C, and DR antigens, and (2) expression of common monocyte/macrophage-associated antigen(s) and uniquely associated antigen(s) selectively expressed on tissue macrophages. These reagents will be useful in delineating human monocyte/macrophage differentiation as well as the immunological functions of mononuclear phagocytes.     

Intracellular turnover of stable and labile soluble liver proteins

The cytosol proteins of mouse or rat liver were separated on the basis of charge and characterized with respect to molecular size, turnover in vivo, and several chemical properties. The basic proteins, which were synthesized and degraded slower than acidic proteins, were generally smaller, as multimers and subunits, than the acidic proteins. Charge and size, therefore, did not appear to be independent characteristics for soluble liver proteins. The amino acid composition of the smaller, basic, stable proteins was very similar to that of the larger, acidic, labile proteins. The charge differences between these two protein fractions could be attributed to the ratio of acidic to basic amino acid residues rather than to carbohydrate, nucleic acid, or phosphate content. The percentage of hydrophobic amino acid residues in the two protein fractions was the same. The acidic labile proteins were more vulnerable to proteolysis and associated with the lysosomal-mitochondrial fractions somewhat more extensively in vitro than the basic, stable proteins. These studies indicate that the information determining the half-lives of soluble enzymes is in the protein moiety of those enzymes and that the factors that correlate with turnover may not be independent variables for soluble mammalian proteins.     

Endocytosis and degradation of native, cathepsin D-degraded, and glutathione-inactivated aldolase by perfused rat liver

The uptake and degradation of 125I-labeled (a) native aldolase, (b) cathepsin D-inactivated aldolase, and (c) aldolase inactivated by oxidized glutathione were studied in perfused rat liver. All three forms of aldolase were removed from the perfusion medium and degraded by the liver, but the uptake of the glutathione-inactivated enzyme (half-life in perfusate = 10 min) was much faster than that of the native enzyme (half-life = 30 min) or the cathepsin-inactivated enzyme (half-life = 42 min). The degradation of the enzyme was almost totally inhibited by leupeptin, indicating that thiol proteinases in lysosomes play an important role in the digestion process. Degradation of native and cathepsin D-inactivated aldolase appeared to be slower than that of the glutathione-inactivated enzyme but studies in which liver was preloaded with aldolase by perfusion at 19 degrees C and then warming to 37 degrees C indicated that the rate of degradation of all three forms was similar. It is concluded that the liver is capable of distinguishing between the glutathione-altered aldolase and native or partially degraded aldolase with regard to endocytosis, but that all three forms are degraded at similar rates once within lysosomes.     

Generation of nonspecific murine cytotoxic T cells in vitro by purified human interleukin 2

Murine spleen cells developed into nonspecific cytotoxic cells within 72 hr of culture in the presence of highly purified sources of human interleukin 2. In whole spleen cell cultures, human interleukin 2 generated effector cells which were Thy 1.2⁺, Lyt 2.2⁺, resistant to γ irradiation (1000 R), and capable of lysing both H-2 compatible and incompatible targets. The effector cells generated in this manner were not restricted to classical natural killer cell-sensitive targets. If thymus-derived cells (T cells) were depleted from the spleen cell population before culture with human interleukin 2, the effector cells generated were enriched in effectors capable of lysing natural killer cell-sensitive targets. Interferon was not produced in interleukin 2-stimulated spleen cell cultures. In addition, heterologous antibody to murine -γ-interferon did not abrogate the generation of cytotoxic cells by human interleukin 2. These and additional data suggest that human interleukin 2 is capable of stimulating γ-irradiation-sensitive Thy 1.2⁺ cell(s) capable of lysing a variety of target cells regardless of inherent sensitivities to classical natural killer cells. Thy 1.2^? cells were also stimulated by human interleukin 2 and lysed only natural killer cell-sensitive targets. Human interleukin 2 caused some Thy 1.2^? cells to become susceptible to lysis by anti-Thy 1.2 serum and complement.     

Role of HLA-DR bearing Langerhans and epidermal indeterminate cells in the in vitro generation of alloreactive cytotoxic T cells in man

Human epidermal cells (EC) act as stimulator cells in the mixed-skin cell lymphocyte culture reaction (MSLR). To analyze the role of human epidermal Langerhans cells (LC) and indeterminate cells (IC), which are the only cells expressing the DR-Ia-like antigens in normal epidermis, in the generation of alloreactive cytotoxic T lymphocytes (CTL) in cell-mediated cytolysis, 18-hr 51Cr-release assays against PBL targets (targets autologous to stimulator EC) were conducted after allogeneic human MSLR. MSLR and CTL assays were conducted with, as stimulator cells, suspensions of normal human EC as controls, and EC after: (1) preincubation with anti-HLA-DR or OKT6 (specific for LC in EC suspension) monoclonal antibodies; (2) panning, a monolayer technique used to deplete EC suspensions in OKT6 or DR-positive cells. The generation of alloreactive CTL was found to occur only after allogeneic MSLR and when targets and stimulator cells were from the same donor; it was reduced by EC incubation: cytotoxic activity 26.66 +/- 3.84 (controls); 8.8 +/- 3.6 and 7.7 +/- 3.7 (EC incubated with OKT6 or anti-DR, respectively); it was reduced or abolished when the EC used in MSLR were depleted in OKT6 or DR-positive cells by panning. These findings demonstrate that human LC and IC are necessary for an optimal in vitro sensitization in MSLR and the subsequent in vitro generation of alloreactive CTL in man.     

Cultivation and characterization of macrophages from murine embryonic skin. Are they Langerhans cells?

We have observed a population of trypsin-resistant adherent cells in long-term primary cultures of murine embryonic skin. These cells were subsequently demonstrated to share a variety of characteristics with cells of the monocyte/macrophage lineage. The trypsin-resistant adherent cells stained positively for nonspecific esterase, exhibited surface receptors for Fc-IgG, and complement components as well as strong phagocytic activity. Additionally, these cells exhibited membrane ATPase enzyme activity and a large proportion of the cells expressed la antigens as detected by cytotoxicity and membrane fluorescence. The possible relationship between these trypsin-resistant adherent cells and Langerhans cells of the skin is discussed.     

T-cell neoplasm induced by subcutaneous transplantation of a plasmacytoma: characterization of tumor cells.

Thymocytes, isolated 6 days following subcutaneous (sc) transplantation of BALB/c MOPC-315 plasmacytoma into F₁ (BALB/c × C57BL) hybrid mice, when injected sc into normal syngeneic mice, caused the development of a solid sc tumor. The cells of the newly developed tumor were of a mixed population of θ⁺F₁ (BALB/c × C57BL) and θ^? BALB/c cells (approximately 1:1), which represents a new type of mixed T cell-plasma cell neoplasm. Efforts were made to isolate the transformed thymocytes (θ⁺) from the plasmacytoma (θ^?) cells in the new tumor, exploiting differences in their surface properties. Treatment of the mixed tumor cell population with peanut agglutinin (PNA) revealed that only the T tumor cells were agglutinated. The agglutinated cells were recovered after dispersing the clumps with d-galactose (0.15 M) and consisted of 95% θ⁺ cells. The PNA-agglutinated cells were found to induce a similar tumor (85% θ⁺ cells) when injected sc into F₁ (BALB/c × C57BL) mice.     

Comparative analysis of the heterogeneity of mononuclear cells present in adult and cord blood by simultaneous examination of multiple phenotypic characteristics

In an attempt to better relate specific membrane characteristics of human adult and cord blood lymphocytes to specific functional activities, the phenotypic differences that exist in these two populations have been examined. Cord blood cells have considerably more spontaneous suppressor cell activity than adult cells. A technique that allows cells to be examined simultaneously for their ability to ingest latex beads, react with specific monoclonal antisera, bind sheep erythrocytes, or react with the Fc portion of IgG was used. As well as assessing fresh populations, phenotypic changes that occur when such cells are held in culture or stimulated with phytohemagglutinin for 3 days were sought. Many differences were found when comparing these mononuclear populations. These included the observations that 12% of adult and 9% of cord blood E-rosette-forming cells ingest latex beads and that 9% of OKT3 reactive cells in both populations did not form E rosettes. In cord blood 58% of T cells that bind OKT8 do not form E rosettes. A similar percentage of cord blood T8-positive cells express a receptor for Fc gamma, such cells being very uncommon in adult blood. Four "monocyte" subpopulations were identified in both samples. One such population (an OKM1- and Fc gamma-positive, nonphagocytic cell) was three times more common in cord blood. In cord blood some OKM1-positive cells also appeared to be simultaneously OKT8 positive. These phenotypic variations forward populations that may be candidates responsible for the functional differences noted in vitro.     

Immunoadjuvant activity of pyran copolymer: I. Evidence for direct stimulation of T-lymphocytes and macrophages

A single injection of pyran copolymer has been shown to greatly increase the number of hemolytic plaque forming cells to sheep erythrocytes (sRBC). Pyran given from 1 day before to 2 days after sRBC inoculation increased both specific activity and plaques/spleen, suggesting that macrophage activation was probably not responsible for the enhancement seen. In addition, pyran given 1 day prior to the primary injection of sRBC was found to increase the secondary response to SRBC given alone. As similar experiments using thymectomized irradiated bone marrow reconstituted mice showed no increase in specific activity following pyran administration, it was unlikely that pyran was acting directly on B cells. Furthermore, experiments measuring the antibody response to Escherichia coli lipopolysaccharide, a thymic independent antigen, pyran did not increase the response to this antigen. In contrast to the above, pyran delayed and depressed cell mediated cytotoxicity to the allogeneic DBA/2 P815 mastocytoma. However, no difference in the titers of cytotoxic antibody against mastocytoma cells was seen between pyran-treated and normal animals. Pyran was mitogenic for spleen cells in vitro. However, following the administration of pyran in vivo, mitogen induced blastogenesis in vitro to PHA and LPS was inhibited and this inhibition was determined to be macrophage-dependent. These results are consistent with a model in which the immunoregulatory effects of pyran act through macrophages and T-lymphocytes.     

Role of proton dissociation in the transport of acidic amino acids by the Ehrlich ascites tumor cells

The pH profile for the uptake of l-glutamic acid by the Ehrlich ascites tumor cell arises largely as a sum of the decline with falling pH of a slow, Na⁺-dependent uptake by System A, and an increasing uptake by Na⁺-independent System L. The latter maximizes at about pH 4.5, following approximately the titration curve of the distal carboxyl group. This shift in route of uptake was verified by (a) a declining Na⁺-dependent component. (b) an almost corresponding decline in the 2-(methylamino)-isobutyric acid-inhibitable component, (c) a rising component inhibited by 2-aminonorbornane-2-carboxylic acid. Other amino acids recognized as principally reactive with Systems A or L yielded corresponding inhibitory effects with some conspicuous exceptions: 2-Aminoisobutyric acid and even glycine become better substrates of System L as the pH is lowered; hence their inhibitory action on glutamic acid uptake is not lost. The above results were characterized by generally consistent relations among the half-saturation concentrations of the interacting amino acids with respect to: their own uptake, their inhibition of the uptake, one by another, and their trans stimulation of exodus, one by another.A small Na⁺-dependent component of uptake retained by l-glutamic acid but not by d-glutamic acid at pH 4.5 is inhibitable by methionine but by neither 2-(methylamino)-isobutyric acid nor the norbornane amino acid. We provisionally identified this component with System ASC, which transports l-glutamine throughout the pH range studied. No transport activity specific to the anionic amino acids was detected, and the unequivocally anionic cysteic acid showed neither significant mediated uptake nor inhibition of the uptake of glutamic acid or of the norbornane amino acid.     

The requirement for biotin and fatty acids in the cytotoxic T-cell response.

Dialyzed fetal calf serum (FCS) was a poor source of serum supplement for in vitro cytotoxic T lymphocyte (CTL) generation. Serum dialysate or biotin fully restored dialyzed FCS to activities comparable to FCS. It was concluded that the active principal in serum dialysate was biotin because its further dialysis was prevented by addition of avidin, a biotin binding protein. Avidin inhibited CTL generation only when added during the early stages of mixed lymphocyte cultures, whereas biotin could restore activity even if added at a later time. When FCS enriched in a fatty acid mixture, or in palmitic acid alone, was used as the serum supplement, avidin-mediated inhibition of CTL generation was markedly reduced. Avidin also inhibited CTL generation in cultures containing killed macrophages as the stimulating cell, and supplemented with Con-A-induced spleen cell supernatant, a source of helper factor(s). These experiments suggest that fatty acid biosynthesis and the attendant synthesis of structural lipids of appropriate fatty acid composition play a prominent role in the generation of CTL     

A multiple plasmid-containing Escherichia coli strain: convenient source of size reference plasmid molecules.

A strain of Escherichia coli is described that contains eight plasmid species ranging in size from 1.36 × 10⁶ to 35.8 × 10⁶ daltons. This strain can be employed as a single source of covalently closed circular deoxyribonucleic acid molecules of different sizes for use as references in agarose gel electrophoretic analysis.     

Mapping unintegrated avian sarcoma virus DNA: termini of linear DNA bear 300 nucleotides present once or twice in two species of circular DNA.

Three major species of viral DNA have been observed in cells infected by retroviruses: a linear, double-stranded copy of a subunit of viral RNA; closed circular DNA; and proviral DNA inserted covalently into the genome of the host cell. We have studied the structures of the unintegrated forms of avian sarcoma virus (ASA) DNA using agarose gel electrophoresis in conjunction with restriction endonucleases and molecular hybridization techniques. The linear duplex DNA is approximately the same length as a subunit of viral RNA (approximately 10 kb) and it bears natural repeats of approximately 300 nucleotides at its termini. The repeats are composed of sequences derived from both the 3' and 5' termini of viral RNA in a manner suggesting that the viral DNA polymerase is transferred twice between templates. Thus the first end begins with a sequence from the 5' terminus of viral RNA and is permuted by about 100 nucleotides with respect to the 3' terminus of viral RNA; the linear DNA terminates with a sequence of about 200 nucleotides derived from the 3' end of viral RNA. We represent this structure, synthesized from right to left, as 3'5'-----3'5'. Two closed circular species of approximately monomeric size have been identified. The less abundant species contain all the sequences identified in linear DNA, including two copies in tandem of the 300 nucleotide 3'5' repeat. The major species lacks about 300 base pairs (bp) mapped to the region of the repeated sequence; thus it presumably contains only a single copy of that sequence. The strategies used to determine these structures involved the assignment of over 20 cleavage sites for restriction endonucleases on the physical maps of ASV DNA. Several strains of ASV were compared with respect to these sites, and the sites have been located in relation to deletions frequently observed in the env and src genes of ASV.     

Malignant transformation-linked imbalance: decreased xanthine oxidase activity in hepatomas.

Xanthine oxidase was decreased 2- to 10-fold in all examined rat hepatomas irrespective of the malignancy; growth rate and degrees of histological differentiation of the neoplasms. The affinity to substrate (KM=6-8 muM) and the pH optimum (8.0) of the liver and hepatoma enzymes were the same. The reprogramming of gene expression, as manifested in the decreased activity of this key purine metabolizing enzyme, appears to be specific to neoplastic transformation. Since glutamine PRPP amidotransferase activity was increased but the opposing enzyme, xanthine oxidase, was decreased in all the hepatomas, the reprogramming of gene expression results in an imbalance that favors synthesis against catabolism. This enzymatic imbalance should confer selective advantages to the cancer cells.     

Sex differences in the activity of mice: modulation by postnatal gonadal hormones

A series of six experiments was performed to examine the influence of postnatal-gonadal-hormone exposure on home-cage activity in Rockland-Swiss albino mice. Intact females were more active than their male counterparts and gonadectomy in adulthood, while reducing levels of the behavior in both sexes, did not eliminate the gender difference. Males that were castrated on the day of birth were more active than animals castrated 5, 10, or 25 days later. Also, females treated with testosterone propionate on the day of birth were less active than oil-treated controls and females exposed to the steroid 10 days after birth. Thus, perinatal exposure to gonadal hormones suppresses adult levels of home-cage activity in mice.     

Effect of acetate on glucose utilization by isolated rat thymocytes

The effects of acetate and ammonium salts on glucose metabolism, aminoisobutyric acid influx, and radioiodinated insulin binding in isolated thymocytes were studied. Acetate in the concentration range, 0.1–30 mm, was found to inhibit basal and insulin-stimulated CO₂ production whereas ammonium chloride at concentrations greater than 0.3 mm was slightly stimulatory. Ammonium salts inhibited glucose incorporation into glycogen and aminoisobutyric acid influx only at high concentration (30 mm). Neither acetate nor ammonium salts had significant effects on glucose incorporation into glycogen or aminoisobutyric acid influx at lower concentrations. No effect on insulin binding was observed. The observation that very low concentrations of acetate can perturb these biological assay systems suggests that other biological functions may be affected by trace amounts of buffer salts carried over from protein isolation steps.     

Analysis of migratory behavior of neural crest and fibroblastic cells in embryonic tissues

The precise migration of neural crest cells is apparently controlled by their environment. We have examined whether the embryonic tissue spaces in which crest cells normally migrate are sufficient to account for the pattern of crest cell distribution and whether other migratory cells could also distribute themselves along these pathways. To this end, we grafted a variety of cell types into the initial crest cell migratory pathway in chicken embryos. These cell types included (a) undifferentiated neural crest cells isolated from cultured neural tubes, intact crest from cranial neural folds, and crest derivatives (pigment cells and spinal ganglia); (b) normal embryonic fibroblastic cells from somite, limb bud, lateral plate, and heart ventricle; and (c) a transformed fibroblastic cell line (Sarcoma 180). Crest cells or their derivatives grafted into the crest migratory pathway all distributed normally, although in contrast to the result when neural tubes were graftedin situ, fewer cells were observed in the epithelium and few or none were localized in the nascent spinal ganglia. Grafted quail somite cells contributed to normal somitic structures and did not migrate extensively in the chicken host. Other fibroblasts did not migrate along cranial or trunk crest pathways, or invade adjacent tissues, but remained intact at the graft site. Sarcoma 180 cells, however, distributed themselves along the normal trunk crest pathway. Cranial and trunk crest cells and crest derivatives grafted ectopically in the limb bud or somite also dispersed, and were found along the ventral migratory pathway. Fibroblastic cells grafted into ectopic sites again remained intact and did not invade host tissue. We conclude (1) that neural crest cells and their derivatives are highly motile and invasive in their normal pathway, as well as in unfamiliar embryonic environments; and (2) that the crest pathway does not act solely to direct neural crest cells, since at least one transformed cell can follow the crest migratory route.     

Cytochrome P-450 and drug metabolism in intestinal villous and crypt cells of rats: effect of dietary iron.

The amino acid sequence of the $\text{Spirulina maxima}$ ferredoxin has been determined. $\text{Spirulina maxima}$ is a blue green algae and is a procaryote. The ferredoxins of the plant-algal type sequenced to date have all been isolated from eucaryotes. The $\text{S. maxima}$ ferredoxin was composed of 98 amino acids arranged in a single polypeptide chain.The sequences of the various procaryote-eucaryote ferredoxins are compared and the differences discussed.