Detection of double target binders at the single cell level: an evaluation of the specificity of NK and MLC-induced NK-like cells

MLC-generated cells were tested on 7 consecutive days in the single cell cytotoxicity assay to determine the kinetics of natural and allospecific killing. Maximum cytotoxicity to the NK-sensitive target, K562, was found on Day 3 of MLC with an increase at that time in both the number of cells binding and the number of cells killing K562. The maximum allospecific response was found on Days 6 and 7 with an increase in cells able to bind and kill the alloantigen-bearing target. To determine whether the anti-K562 and allospecific killing were mediated by the same effector cells or different cell populations, both targets were tested simultaneously in the single cell assay. At no time during the 7 days were cells detected capable of simultaneously binding both K562 and allospecific targets. These data indicate that there are two different cell populations responsible for allospecific cytotoxicity and MLC-induced NK-like cytotoxicity. The cytotoxic specificity of unstimulated and MLC-generated NK-like cells was also investigated. When two different NK-sensitive targets (e.g., K562 and MOLT-4) were tested together in the single cell assay, there was no concurrent binding of targets by either fresh PBL prior to MLC stimulation or Day 3 MLC-generated cells. When unstimulated effector cells were enriched for NK activity by Percoll density gradient centrifugation, only a small number of effector cells simultaneously binding two different NK-sensitive targets was detected in the single cell assay. These results imply that the NK cell population is heterogeneous and composed of subpopulations recognizing diverse target specificities.     

Regulation of acetylcholinesterase activity by retinoic acid in a human neuroblastoma cell line

The ability of retinoic acid (RA) to modulate acetylcholinesterase (AChE) activity in a human neuroblastoma cell line (LN-N-5) was examined. The specific activity of AChE was significantly increased 3 days after exposure of LA-N-5 to RA and reached its maximum values after 9 or more days of culturing. Dose-response experiments demonstrated that large increases of AChE occurred at RA concentrations between 10(-7) and 10(-6) M with maximum AChE values detected at 10(-6)-10(-5) M. Increased AChE activity paralleled neurite outgrowth in LA-N-5 cultures. These findings demonstrate that RA can regulate specific AChE activity in human neuroblastoma cells in a manner consistent with neuronal maturation.     

Immunoglobulin properties of Epstein-Barr virus transformed human umbilical cord and adult peripheral blood lymphocytes

We have observed that a subset of E+ cells bind human monomeric IgG (FcR-IgG). In the present study, we have separated the E+, FcR-IgG+ cells by flow cytometry and tested their NK activity against the tumor KS62 using the chromium-release assay. Virtually all the NK activity residing within the E+ subset was mediated by E+, FcR-IgG+ cells. These findings are discussed in relation to the cellular lineage of the human NK cell.     

Membrane defects of the tolerant B cell. I. Failure of antigen-induced capping.

In order to study the membrane function of tolerant B antigen-binding cells, tolerance to the trinitrophenyl (TNP) determinant was induced in mice by injecting the reactive form of the hapten, trinitrobenzene sulfonic acid (TNBS). By appropriate transfer experiments, Fidler and Golub (J. Immunol.112, 1891, 1974) had previously shown that this form of tolerance is a B-cell property, induced and expressed in the absence of T cells. Hapten inhibition demonstrated the TNP-specificity of receptors on TNP-donkey erythrocyte(TNP-D)-binding cells in tolerant and nontolerant mice. About 88% of these cells were B cells by immunofluorescence, and the remainder were T cells. In the tolerant mice, challenge with TNP-sheep erythrocytes failed to expand the TNP-binding population, but sheep erythrocyte binders and anti-sheep plaque-forming cells expanded normally. Despite little or no change in TNP-binding cell numbers after tolerance induction, the TNP-binding cells of tolerant animals could not cap their receptors, in contrast to the sheep erythrocyte-binding cells from the same animals which capped normally. Although there is no anti-TNP plaque-forming cell response when tolerogen and immunogen are given simultaneously, capping failure is not evident until 2–4 days after tolerogen exposure. By Day 7, substantial recovery of immune responsiveness had occurred, yet even 12 months after a single dose of tolerogen there was no restoration of capping. Thus despite the association of both capping failure and unresponsiveness with tolerogen exposure, these lymphocyte functional defects appeared not to be causally related.     

Immunological aspects of retinoids in humans. II. Retinoic acid enhances induction of hemolytic plaque-forming cells

The effects of retinoic acid (RA) on the induction of antibody-producing cells from human tonsillar lymphocytes sensitized to sheep erythrocytes (SRBC) have been evaluated. Our results indicated that 10(-5) to 10(-7) M RA caused up to a three-fold increase in the number of plaque-forming cells (PFC) and a qualitative increase in the size of the plaques during the induction of PFC in 5- to 7-day cultures. Enhancement also occurred when tonsil cells were preincubated with RA for 24 hr and then washed, or when RA was added any time in the first 4 days after initiation of the culture. When T- and B-cell fractions were pretreated with RA for 24 hr, washed, and recombined with SRBC, RA-induced augmentation of PFC occurred only in conjunction with RA treatment of the B-cell fraction. Pretreatment of the T-cell fraction had no effect on PFC induction or on the RA-enhanced response when the B-cell fraction was simultaneously treated with RA. Other experiments suggested that RA did not modulate PFC induction by influencing regulatory functions of adherent accessory cells. Our study demonstrates that RA can enhance human antibody responses and shows that this effect is not caused by increased activity of T cells or adherent accessory cells, but is instead the result of a direct effect of RA on B-cell populations.     

Membrane defects in the tolerant B cell. II. Mechanism of capping failure and reversal by antigen exposure.

The demonstration that TNP-binding B lymphocytes from animals whose B cells have been rendered tolerant to TNP by trinitrobenzene sulfonic acid cannot undergo antigen-induced capping of their TNP receptors for at least a year despite recovery of immune responsiveness has led to a search for the mechanism of the capping failure. Microtubule-dependent membrane “locking” analogous to that induced by concanavalin A appears to afflict the tolerant B cells, in that capping TNP receptors is restored after exposure to 10^?4M colchicine or overnight incubation at 4 °C. Assignment of the defect to the cytoskeleton rather than the receptors themselves is also supported by the observations that enzymatic stripping and regrowth of receptors does not unlock the cell and that non-Ig membrane molecules recognized by antilymphocyte serum also cannot be capped on the tolerant cells. Cells which have remained locked for 4 days to 8 months after a single tolerogen exposure become unlocked 4 days after immunogen is given. Four days after immunogen, tolerogen fails to lock the membranes of TNP-binding cells. These results suggest that tolerogen contact interferes in a much broader range of functions in the TNP-binding cell than those which affect the immune response. Among these effects is a remarkably stable “locked” configuration of the cytoskeleton which is independent of immune responsiveness or receptor turnover, but which can be reversed by exposure to immunogen whether or not an immune response ensues.     

Enhanced sensitivity of trisomy 21 monocytes to the maturation-inhibiting effect ot interferon

Cultured peripheral blood monocytes from subjects with trisomy 21 (Down Syndrome) demonstrated a 3.7-fold enhanced sensitivity to the maturation-inhibiting effect of leukocyte interferon. This increased sensitivity is considered to be the result of the presence of an increased concentration of the interferon receptor, which is controlled by the IfRec locus on human chromosome 21, on the surface of the trisomic monocytes. Since macrophages are important components of many immune processes and interferon is itself a product of and has regulatory functions in immune reactions, the enhanced sensitivity of trisomic monocytes to interferon may be a factor leading, paradoxically, to the greater susceptibility of trisomic individuals to viral and bacterial infections.     

Estrogens before birth and development of sex-related reproductive traits in the female guinea pig

Influences of estrogens on the differentiation of psychosexual traits in the female guinea pig were studied. Pregnant animals were injected intramuscularly with either 1, 2, or 3.3 micrograms estradiol benzoate (EB) or with 1 or 3 micrograms diethylstilbestrol dipropionate (DESDP). Injections were started on the 29th day of pregnancy, given daily for 6 days, and continued every other day until parturition. Female offspring were evaluated for onset of puberty, ovarian function, and lordosis and mounting behavior in adulthood. Prenatal treatment with 3 micrograms DESDP caused delayed puberty, impaired ovarian function, reduced responsiveness of lordosis to EB and P in adulthood (defeminization), augmented mounting in the absence of hormones (masculinization), and reduced responsiveness of mounting to exogenous EB and P in adulthood (defeminization). Prenatal treatment with 1 microgram DESDP produced similar but less pronounced effects. Prenatal treatment with 3.3 micrograms EB also caused a delay in puberty. However, responsiveness of lordosis to EB and P in adulthood was enhanced by treatment with either 1 or 3.3 micrograms EB prenatally. Further, neither mounting in the absence of hormones nor mounting in response to EB and P in adulthood were affected in any measurable way by any prenatal treatment with EB. These results show that estrogens can have masculinizing and defeminizing effects on sexually dimorphic reproductive traits in guinea pigs. The failure of EB to duplicate or parallel the effects of DESDP is not completely understood at this time, but it may indicate that less of the active substance reaches the target tissues following maternal and placental metabolism of EB than of DESDP.     

Inhibition of natural killer cell lysis by natural killer-inhibitory substance: mechanism of suppression

The mechanism of suppression of NK-mediated lysis by a soluble product of peritoneal cells (NK-IS, natural killer-inhibitory substance) was investigated. Pretreatment of effector cells resulted in depressed NK lysis while pretreatment of targets had no effect, indicating suppression is due to alterations in effector cell function rather than changes in target cells. NK-IS had no effect on the formation of conjugates between effectors and NK-susceptible targets. When NK-IS was added to effector-target cell mixtures after the binding step had been successfully completed, ensuing lysis was significantly depressed, confirming that NK-IS inhibited a postbinding lytic event. The degree of suppression caused by NK-IS was directly related to the duration of exposure to the inhibitory molecule. In addition, a preliminary temperature-dependent step of binding to and/or intracellular entry of NK-IS into effectors is required before suppression can occur. NK-IS prevents the activation of NK cell lysis by interferon and Corynebacterium parvum and effectively inhibits lysis mediated by already activated effectors. The potent suppression of NK lysis and prevention of interferon and C. parvum-mediated activation of NK lysis by a soluble product of peritoneal cells may explain the extremely low level of NK effector cell function within the peritoneal cavity.     

Cerebral endothelial cell culture. I. The presence of beta 2 and alpha 2-adrenergic receptors linked to adenylate cyclase activity

Cultured endothelial cells derived from cerebral microvessels separated from 2-day-old rat brain contain a specific beta 2 and alpha 2-adrenergic sensitive adenylate cyclase (AC). Among the various tested hormones, PGE1 and PGE2 were found to be the most potent activators, while adenosine, angiotensin I and II, gamma-aminobutyric acid and vasoactive intestinal peptide inhibited the enzyme activity. However, acetylcholine, histamine, serotonin, glycine, glutamine, bradykinin, neurotensin and vasopressin (Lysine and Arginine) had no effect on the adenylate cyclase activity in this model. The susceptibility of the cerebrovascular endothelial AC system to the vasoactive substances as well as presence of beta 2 and alpha 2-type adrenergic receptors in the cultured endothelium provides additional support for the proposed endothelial involvement in the regulation of cerebrovascular permeability and blood flow.     

The induction of suppressor T cells by lipopolysaccharide in human peripheral blood lymphocyte cultures in the presence of fetal calf serum

Alveolar macrophages (AM) were collected by repeated endobronchial lavage from mice, rats, guinea pigs, and rabbits, and titrated into cultures of mitogen-stimulated syngeneic or autochthonous lymphocytes. Significant species differences were detected in regard to AM activity in the cultures. AM from guinea pigs and mice stimulated PHA-induced lymphoproliferation, while those from rats and rabbits were inhibitory; blood or peritoneal macrophages were not inhibitory in any of the species examined.     

Terminal deoxynucleotidyl transferase activity in a cell line (molt-4) derived from the peripheral blood of a patient with acute lymphoblastic leukemia

A terminal deoxynucleotidyl transferase having a sedimentation coefficient of 3–4S has been found associated with the chromatin from a cell line (Molt-4) derived from the peripheral blood of a patient with acute lymphoblastic leukemia.     

Effects of retinoic acid (RA) on the growth and phenotypic expression of several human neuroblastoma cell lines

It has been shown that retinoic acid (RA) can promote morphologic differentiation and inhibit the growth of a human neuroblastoma cell line, LA-N-1. The present study tests the histological generality of these phenomena by determining the effects of RA on seven other human neuroblastoma cell lines. Results show that RA strongly inhibited anchorage-dependent growth and induced morphologic alterations in six of seven of the cell lines. These alterations included morphologic differentiation as evidenced by formation of neurite extensions in four of the lines, cellular enlargement and vacuolization in one culture, and formation of large, flattened epithelial or fibroblastic-like cells in another culture. Although one cell line was relatively insensitive to the effects of RA in monolayer culture, all seven were strongly inhibited by RA in soft agar assays. Cellular RA-binding proteins were detected in 2/2 lines tested. These findings suggest that, as a histological group, human neuroblastoma cells are extremely sensitive to RA-induced growth inhibition and morphological alterations generally associated with reduced expression of the malignant phenotype of this type of cancer.     

Activation and mechanism of action of suppressor macrophages

Intravenous administration of Corynebacterium parvum to alloimmunized mice activates splenic suppressor macrophages that effectively curtail primary and secondary generation of cytotoxic T lymphocytes (CTLs) in vitro. CTL generation was significantly inhibited in suppressed primary cultures by Day 3, the earliest time point that activity is first detected in control cultures. Suppressor macrophages had to be present during the first 24–48 hr of culture to effectively curtail the generation of CTLs. However, if suppressor macrophages were reactivated by 48-hr in vitro culture and then added to primary sensitizations that had been initiated 48 hr previously, they were capable of significant suppression. Suppressor cells produced a soluble factor that mediated the inhibition of CTL generation. The production or action of this factor could not be counteracted by indomethacin.     

Rapid kinetics of lysis in human natural cell-mediated cytotoxicity: Some implications

The entire lytic process of natural cell-mediated cytotoxicity against sensitive target cells can occur rapidly, within minutes. This was demonstrated by ⁵¹chromium release and in single-cell assays. At the cellular level, most of the target cell lysis occurred within 15–30 min after binding to effector cells. The enriched natural killer cell subpopulation of lymphocytes obtained by Percoll density gradient centrifugation (containing >70% large granular lymphocytes (LGL)) was the most rapidly lytic population by ⁵¹chromium release. However, in the single-cell assay, the rate of lysis of bound target cells was quite similar for the LGL-enriched effector subpopulation and the higher density subpopulation of effector cells recognized previously. Both the light and dense effector cells contained similar numbers of target binding cells. Therefore, that the light subpopulation effected lysis more rapidly and to a greater extent than the dense subpopulation suggested that the low-density effector cells probably recycled more rapidly than those of higher density. This was corroborated by the finding that when conjugates were formed at 29 °C for the single-cell assay, a significant number of dead unconjugated targets could be observed only on the slides made with the LGL-enriched effector cells but not on those made with dense effector cell. Lysis continued to increase in the chromium-release assay probably because of recycling, recruitment, and/or heterogeneity of the effector cells, and/or because of heterogeneity or delayed death of the target cells.     

Effects of 2-pyridine aldoxime methochloride on cerebral acetylcholinesterase activity and respiration in cats poisoned with sarin

2-pyridine aldoxime methochloride (2-PAM Cl) has been reported to be only marginally effective in reactivating brain acetylcholinesterase (AChE) in animals poisoned with organophosphorus anticholinesterase compounds. In the present studies, an effort was made to obtain additional information on the effects of 2-PAM Cl on AChE activity of the cerebral cortex and respiration in cats intoxicated intravenously with either 1.8 × LD50 (27 μg/kg) or 3 × LD50 (45 μg/kg) of isopropyl methylphosphonofluoridate (Sarin). Following poisoning by 1.8 × LD50 of Sarin, injection of 15 mg/kg of 2-PAM Cl into the common carotid artery in a volume of 5 ml/kg of saline, pH 7.3, affected AChE activity in the cerebral cortex. The enzymatic levels in the poisoned, oxime-treated cats were significantly higher than those of the poisoned, untreated animals. In other experiments, the time for recovery of respiration from the effects of 3 × LD50 of Sarin was reduced from an average of 147 min in untreated animals to within 2 min after oxime administration. Under the conditions of these experiments, 2-PAM Cl rapidly restored respiration and reactivated some AChE in the cerebral cortex of cats intoxicated with Sarin.     

The relationship between the osmotic fragility of human erythrocytes and cell age

Erythrocytes in a normal blood sample are hemolyzed over a range of hypotonic salt concentrations. In order to investigate the relationship between the distribution of osmotic fragilities and the distribution of cellular ages, the osmotic fragility has been compared with three indices of cellular age. The activity of glutamic-oxalacetic transaminase (GOT) and the percentage hemoglobin A_1C were measured in samples hemolyzed in different hypotonic salt concentrations. The osmotic fragility curve was also obtained for cells of different density separated by centrifugation. These experiments indicate that the fragility distribution is not an accurate reflection of the distribution of cellular ages. The mean fragility for older cells is higher than that of younger cells. However, cellular aging does not produce a gradual increase in osmotic fragility. Instead, it seems to produce changes which can both increase and decrease the fragility, resulting in a broader distribution of fragilities with some of the older cells actually less fragile than the younger ones.     

Differential effects of positive and negative proliferative stimuli on murine cytolytic and helper T-cell clones

Studies were performed to determine whether substances could be identified which exhibited differential regulatory effects--either positive or negative--on the growth of murine alloreactive cytolytic (Tc) and helper (Th) cloned T-cell lines. The following lines of evidence suggested that Tc and Th proliferate in response to the same growth factor (GF). (1) When GF-containing fluids from cultures of phorbol myristic acetate (PMA)-activated EL4 thymoma were fractionated by a variety of biochemical techniques. Tc and Th eluted together. (2) Absorption of GF-containing supernatants with either cloned Tc or cloned Th depleted GF activity for each to a similar extent, and GF eluted from either Tc or Th to which it had adsorbed supported the proliferation of Tc and Th equally well. (3) Lectin-depleted supernatants from cultures of concanavalin A (Con A)-activated Th stimulated the proliferation of Th as well as Tc. (4) Recombinant human interleukin (IL-2) supported the growth of Tc and Th with equal efficiency. On the other hand, the following observations indicated that Tc and Th differed in their responses to inhibitors of GF-driven proliferation. (1) Con A at greater than or equal to 0.3 micrograms/ml inhibited the GF-driven proliferation of each of three Th lines but not either of two Tc lines. To the contrary, Con A enhanced GF-dependent proliferation of Tc. (2) Like Con A, allogeneic splenocytes selectively depressed GF-driven proliferation of Th but not Tc. (3) A substance generated during the acid elution of GF from cells, possibly a modified fetal calf serum component, greatly reduced the GF-driven proliferation of Tc but not Th. These results suggest that differential control of the proliferation of Tc and Th in cellular immune responses may be achieved via negative regulatory signals and raise the possibility that substances which can selectively depress the proliferation of specific T-cell subsets might be found which would be of therapeutic value.     

Associated effects of sodium butyrate on histone acetylation and estrogen receptor in the human breast cancer cell line MCF-7

Sodium butyrate at a concentration of 5mM causes significant hyperacetylation of the core histones in the human breast cancer cell line MCF-7. Histone hyperacetylation was achieved in rapidly-growing cells at 40% confluency after 24 hours in 5mM sodium butyrate. More nearly confluent cells did not reach as high a level of histone hyperacetylation. Upon assaying the estrogen receptors, both cytosolic and KCl-extractable nuclear, we found that butyrate treatment had lowered the estrogen receptor levels in both compartments. To our knowledge this is the first report of an effect of sodium butyrate on estrogen receptor levels.