Monocyte-independent interleukin-2 production and proliferation of human T cells in response to murine hybridomas expressing the OKT3 monoclonal antibody: interleukin-1 is not required for T-cell proliferation

We report a new, monocyte-independent system for the induction of activation and proliferation of human T cells in response to murine hybridomas expressing the OKT3 monoclonal antibody (OKT3 hybridomas). Incubation of nylon-wool-nonadherent (NA) lymphocytes or purified T cells with OKT3 hybridomas resulted in interleukin-2 (IL-2) production, expression of IL-2 receptor, modulation of the CD3 antigen, and proliferation. In contrast, murine hybridomas (OKT4, OKT8, anti-HLA-DR, and others) expressing monoclonal antibodies (mAb) other than OKT3 did not induce T-cell activation and proliferation. T cells did not respond to OKT3 mAb alone. OKT3 hybridomas alone did not produce interleukin-1 (IL-1) or other soluble factors that might be involved in the induction of IL-2 production by T cells, and they did not contain membrane-bound IL-1. In addition, IL-1 activity was not detected in cultures of NA-lymphocytes and OKT3 hybridomas, clearly demonstrating that IL-1 was not required, at least in this system, for T-cell activation and proliferation. Direct cell-cell contact between T cells and OKT3 hybridomas was required for IL-2 production. Thirty to fifty percent of T cells formed conjugates with the OKT3 hybridomas but not with the OKT4 or OKT8 hybridomas. Both conjugate formation and IL-2 production were significantly inhibited by the OKT3 mAb and by the anti-LFA-1 mAb. The cells responsible for IL-2 production were found to be of the T3+ T4+ T8- Leu 7- Leu 11- phenotype. IL-2 activity produced by NA-lymphocytes in response to OKT3 hybridomas became detectable as early as 1 hr and reached a maximum by 8 hr, preceding IL-2 receptor expression, modulation of the CD3 antigen, and [3H]thymidine incorporation of T cells. T cells produced higher concentrations of IL-2 in response to OKT3 hybridomas than in response to equal numbers of monocytes and OKT3 mAb. Addition of monocytes to cultures of T cells and OKT3 hybridomas resulted in suppression of IL-2 production in a concentration-dependent manner, suggesting that monocytes regulate the levels of IL-2 production. This monocyte-independent system may be useful for further dissection of T-cell activation and proliferation and its regulation by monocytes.     

Generation of activated killer (AK) cells by recombinant interleukin 2 (rIL 2) in collaboration with interferon-gamma (IFN-gamma)

Activation of human peripheral blood mononuclear cells (PBMC) by interleukin 2 (IL 2) and the role of interferon-gamma (IFN-gamma) in the IL 2-induced activation were investigated. Activated killer (AK) cells against NK-resistant tumor cell lines were induced in the medium containing recombinant IL 2 (rIL 2) and autologous serum without any other stimulating agents. AK activity was induced by doses of rIL 2 as low as 3 U/ml, and reached a maximum at 10(3) U/ml. Incubation of PBMC with rIL 2 resulted in IFN-gamma production and augmented NK activity after 1 day of culture, and in induction of AK cells and proliferative response after 2 days of culture. These results suggested that endogenous IFN-gamma was required for rIL 2-induction of AK cells and proliferative response. To prove this, PBMC were cultured with rIL 2 and rIFN-gamma or were pretreated with rIFN-gamma before culture with rIL 2. Both rIFN-gamma treatments of PBMC augmented rIL 2-induced AK activity and proliferative response. rIL 2-induced IFN-gamma production was also enhanced by the rIFN-gamma pretreatment of PBMC. The addition of anti-IFN-gamma antibody to rIL 2 cultures abrogated the rIL 2-induced NK augmentation, AK generation, and proliferative response in proportion to the decreased amounts of endogenous IFN-gamma detectable in culture. rIFN-gamma and/or rIL 2 cultures of PBMC increased Tac antigen expression on cell surfaces as measured by flow cytometry. Enhanced Tac expression by rIL 2 was abrogated by adding anti-IFN-gamma antibody. These data indicate that: 1) AK generation and IFN-gamma production are mediated by IL 2, and 2) IFN-gamma production may be required for IL 2 induction of AK cells and proliferative response. These finding are consistent with the hypothesis that AK generation involves a collaboration between IL 2 and IFN-gamma, in which IL 2 stimulates PBMC to produce IFN-gamma, which in turn acts as a differentiation signal that may be involved in the IL 2-initiated AK generation and proliferative response.     

Wavelength-dependence of the relative rate constants for the main geometric and structural photoisomerization of bilirubin IX alpha bound to human serum albumin. Demonstration of green light at 510 nm as the most effective wavelength in photochemical changes from (ZZ)-bilirubin IX alpha to (EZ)-cyclobilirubin IX alpha via (EZ)-bilirubin.

The kinetics for the quantitatively important reaction: (Formula: see text) that is, the photochemical interconversion between bilirubin and its geometric and structural photoisomers bound to human serum albumin in aqueous solution when various wavelengths of monochromatic light were used, were assayed by h.p.l.c. In order to clarify the wavelength-dependence of the relative rate constants in the individual steps, a light-source with a half-bandwidth of 10 nm was used at increments of 20 nm, in the range from 410 nm to 550 nm. We describe for the first time studies on the wavelength-dependence of rate constants in geometric and structural photoisomerization reactions in vitro of (ZZ)-bilirubin or (EZ)-bilirubin bound to human serum albumin, especially the relative rate constants of cyclization of (EZ)-bilirubin into (EZ)-cyclobilirubin. Because studies in vitro have demonstrated that the wavelengths from 350 to 450 nm are mutagenic, the results obtained indicated that the safest and ideal light-source for phototherapy is green light of 510 nm, which keeps (ZE)-bilirubin concentrations as low as possible, as shown by a maximal value of k2 at 510 nm and a relatively low value of k1 at 510 nm. This light-source still ensures the substantial absorption of (ZZ)-bilirubin, which is the precursor of (EZ)-bilirubin, the intermediate in (EZ)-cyclobilirubin formation and, furthermore, as shown by the maximal value of k5 and a considerable value of k4 at 510 nm, promotes the cyclization of (EZ)-bilirubin derived from (ZZ)-bilirubin even though k3 at 510 nm also shows a peak value.     

Haemolysis of rabbit erythrocytes by a lectin from the seeds of<Emphasis Type="Italic">Croton tiglium</Emphasis>

The kinetics of haemolysis of rabbit erythrocytes byCroton tiglium lectin was studied as a function of concentration of the lectin and erythrocytes. The length of the prelytic period decreased with increasing lectin concentrations, indicating that the secondary events at the membrane which follow the binding of the lectin to cell surface carbohydrate receptors are accelerated at higher surface concentrations of the lectin. The rate or extent of haemolysis was not affected by the inclusion of ions like K⁺, Ca²⁺ and Mg²⁺ in the medium or by the substitution of ionic medium by a non-ionic medium. The inhibition of haemagglutination and haemolysis of rabbit red cells byCroton tiglium lectin by antilectin rabbit serum was observed. A possible mechanism of haemolysis by the lectin is discussed.     

Plant lectins specific for N-acetyl-β-D-galactosamine

N-Acetyl-D-galactosamine in β-linkage being ubiquitous in cell surface glycoproteins, their interaction with lectins specific for this sugar moiety may be a significant event in cell adhesion phenomena. This article discusses the common β-N-acetyl galactosamine-specific lectins, with particular stress on the lectin from winged beans (Psophocarpus tetragonolobus).     

Demonstration of an “Excitation-Contraction Recoupling” Mechanism in Mammalian Ventricular Myocardium

A PROCESS called “excitation-contraction coupling” has been generally accepted to take place only in the direction of excitation to contraction. Through this mechanism a propagated action potential initiates an active state in skeletal or cardiac muscle and the muscle contracts. We propose that, in the mammalian ventricular myocardium at least, the process is not unidirectional and an important reverse coupling between the contractile system and the excitable plasma membrane has been overlooked. Through this feedback interaction the mode of contraction (that is, isotonic or isometric) not only determines the instantaneous electrical state of the plasma membrane, but also influences the mechanical events of the subsequent beats. Thus when Kaufmann et al.¹ recorded intracellular action potentials from cat papillary muscle, the time course of the repolarization was altered depending on the mode of contraction. Some kind of contraction-excitation feedback has also been suggested by Stauch² and Lab^3,4. They showed a difference in the shape of the monophasic action potential, as recorded by a suction electrode, when comparing isotonic and isovolumic contraction of the intact ventricle. But their experimental conditions did not allow satisfactory analysis of the phenomenon.     

Function of the <Emphasis Type="Italic">rel</Emphasis> Gene in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Sokawa et al. suggest that rel^- strains of Escherichia coli possess abnormal protein synthesizing machinery, which cannot carry out normal protein synthesis when the supply of amino-acids is limited.     

“Pleiotypic Response”

A hypothesis has been developed to relate stringent control in bacteria to a set of interactions involved in the regulation of growth of transformed and untransformed mammalian cells.