Expression cloning of a human IL-12 receptor component. A new member of the cytokine receptor superfamily with strong homology to gp130.

A cDNA encoding a human IL-12R subunit was isolated by expression cloning. This subunit is a 662 amino acid type I transmembrane protein with an extracellular domain of 516 amino acids and a cytoplasmic domain of 91 amino acids. It is a member of the hemopoietin receptor superfamily and is most closely related over its entire length to gp130 and the receptors for granulocyte-CSF (G-CSF) and leukemia-inhibitory factor. When expressed in COS cells, this IL-12R subunit binds both human and murine IL-12 with an apparent affinity of 2 to 5 nM. The transfected COS cells express both monomers and disulfide-linked dimers or oligomers of the IL-12R subunit on their surface. However, unlike the IL-6-induced dimerization of gp130, the oligomerization of the IL-12R subunit is not dependent on binding of IL-12. Only the IL-12R subunit dimers/oligomers but not the monomers bind IL-12 with an affinity of 2 to 5 nM. A polyclonal antiserum raised against this receptor subunit specifically inhibits IL-12-induced proliferation of PHA-activated PBMC. The data are consistent with the hypotheses that 1) a dimer/oligomer of the cloned IL-12R subunit (IL-12R-beta) represents the low affinity IL-12 binding site identified on human lymphoblasts, 2) the cloned receptor subunit is involved in IL-12 signal transduction, and 3) an additional, as of yet unidentified subunit is required to generate a high affinity IL-12R complex.

Synergistic effects of interleukin 4 and interleukin 12 on NK cell proliferation.

Our previous studies have demonstrated that interleukin12 (IL-12) (cytotoxic lymphocyte maturation factor/NK cell stimulatory factor) and IL-7 alone have the ability to generate high LAK activity and low proliferative activity in CD56+ NK cells. This study was undertaken to examine the influence of IL-4 on the IL-12-induced activation of CD56+ NK cells. IL-4 did not affect the IL-12-induced generation of LAK activity in CD56+ cells, in contrast to an inhibition of IL-2 and IL-7-induced LAK activity. Most interestingly, the combination of IL-4 and IL-12 resulted in a synergistic proliferative activity (8-fold) in the CD56+ NK cells, and a marked increase in the cell yield at day 5 was detected. Furthermore, the potent effect of IL-4 on IL-12-induced proliferation was restricted to the CD56+ NK cells, as CD56- cell populations were found unresponsive to the combination of IL-4 and IL-12. Furthermore, IL-4 induced a slight increase in the IL-12-stimulated TNF production. IL-12 enhanced the IL-12 receptor (R) expression and IL-4R expression in the CD56+ NK cells. Combined treatment with IL-12 and IL-4 further enhanced the IL-12R expression, most prominently in the CD56+, CD16- NK subpopulation. The increased IL-4R expression induced by IL-12 and the increased IL-12R expression induced by IL-4 may explain the synergistic proliferative activity detected in response to IL-12 and IL-4. IL-4 seems to possess unique stimulatory properties towards resting CD56+ NK cells, when used as a costimulus with IL-12.

Modification of the redox state of cytochrome c oxidase of rice due to certain stress treatments.

The redox state of cytochrome alpha 3 during in situ respiration of leaves of 20-day-old rice seedlings was assessed by in vivo aerobic assay of nitrate reductase, after 1 min exposure to carbon monoxide. Different stress treatments like water and salt stresses, disintegration of leaf tissues and darkness modified the redox state of cytochrome c oxidase. The dark treatment altered the redox state of cytochrome oxidase from reduced to the oxidized state, as judged by its reaction with CO in CO-sensitive rice cultivar. The water and salt stresses as well as the disintegration of leaf tissue on the contrary altered cytochrome oxidase from the oxidized to its reduced state in CO-insensitive cultivars; probably by changing the cellular integrity, turgidity and structure of mitochondrial membrane, and also due to decreased mitochondrial energization.

IL-12 receptor. I. Characterization of the receptor on phytohemagglutinin-activated human lymphoblasts.

IL-12 is a 75-kDa heterodimeric cytokine composed of disulfide-bonded 35-kDa and 40-kDa subunits. Included among the biologic activities mediated by IL-12 is induction of proliferation of PHA-activated human PBL. The concentration of IL-12 required to stimulate maximum proliferation of PHA-activated lymphoblasts is 50 to 100 pM. In this study, highly purified 125I-labeled IL-12 (7 to 15 microCi/microgram; 50 to 100% bioactive) was used to characterize the receptor for IL-12 on 4-day PHA-activated lymphoblasts. The binding of 125I-labeled IL-12 to PHA-activated lymphoblasts was saturable and specific because the binding of radiolabeled ligand was only inhibited by IL-12 and not by other cytokines. The kinetics of [125I]IL-12 binding to PHA-activated lymphoblasts was rapid at both 4 degrees C and 22 degrees C; reaching equilibrium within 60 min. At 22 degrees C, the rate of dissociation of [125I]IL-12 was slow in the absence of competing IL-12 (t1/2 = 5.9 h) and more rapid in the presence of 25 nM competing IL-12 (t1/2 = 2.5 h). The kinetically derived equilibrium dissociation constant ranged from 10 to 83 pM. Analysis of steady state binding data by the method of Scatchard identified a single binding site with an apparent equilibrium dissociation constant of 100 to 600 pM and 1000 to 9000 sites/lymphoblast. The equilibrium dissociation constant for competing ligands and sites per cell calculated from unlabeled IL-12 competition experiments ranged from 164 to 315 pM and 1067 to 3336, respectively, which is in good agreement with the values determined from steady state binding. The variations in KD and sites per cell were dependent on the individual preparations of lymphoblasts. Although the steady state binding data were consistent with a single class of high affinity binding sites, the kinetic dissociation data indicates a cooperative interaction between receptors on PHA-activated lymphoblasts. Affinity cross-linking of surface bound [125I]IL-12 to PHA-activated lymphoblasts at 4 degrees C identified a major complex of approximately 210 to 280 kDa. Anti-IL-12 antibodies also immunoprecipitated a complex of approximately 210 to 280 kDa that was produced by cross-linking unlabeled IL-12 to 125I-labeled lymphoblast cell-surface proteins. Cleavage of this complex with reducing agent identified one radiolabeled protein of approximately 110 kDa. These data suggest that the IL-12 binding site on PHA-activated lymphoblasts may be composed of a single protein of approximately 110 kDa.(ABSTRACT TRUNCATED AT 400 WORDS)

IL-12 receptor. II. Distribution and regulation of receptor expression.

IL-12 is a heterodimeric lymphokine that induces IFN-gamma production by resting PBMC, enhances the lytic activity of NK/lymphokine activated killer cells, and causes the proliferation of activated T cells and NK cells. In this report, we have investigated the expression of IL-12R on mitogen- and IL-2-activated PBMC or tonsillar lymphocytes as well as on a variety of cell lines. The results of radiolabeled IL-12-binding assays indicated that high affinity IL-12R are present on PBMC activated by various T cell mitogens or by IL-2. High affinity IL-12R were also found to be expressed constitutively on a transformed marmoset NK-like cell line HVS.SILVA 40. At the time of peak IL-12R expression, mitogen- or IL-2-activated cells displayed approximately 1000 to 9000 IL-12 binding sites/cell with an apparent Kd of 100 to 900 pM. Kinetic studies revealed that maximum expression of IL-12R occurred earlier on PHA-activated PBMC as compared with PBMC activated by IL-2, and that expression of IL-12R on these cells correlated with their ability to proliferate in response to IL-12. Although IL-2 could up-regulate IL-12R expression on resting PBMC, the ability of mitogen-activated PBMC to up-regulate IL-12R was found to be independent of IL-2. Analysis of IL-12R expression by flow cytometry revealed that receptors for IL-12 are present on activated T cells of both the CD4+ and CD8+ subsets and on activated CD56+ NK cells. In contrast, neither resting PBMC or tonsillar B cells nor tonsillar B cells activated by anti-IgM/Dx, anti-IgM/Dx + IL-2, or SAC + IL-2 displayed IL-12R detectable by flow cytometry or by the radiolabeled IL-12-binding assay. In summary, these results indicate that activation of T cells or NK cells results in up-regulation of IL-12R expression; on the other hand, B cell activation, at least under some circumstances, appears not to be associated with enhanced expression of IL-12R.

Differential carbon monoxide sensitivity of cytochrome C oxidase in the leaves of c(3) and c(4) plants.

CO sensitivity of cytochrome a(3) in the leaves of a number of C(3) and C(4) plants was monitored by the nitrate reductase assay under differing CO to O(2) ratios. All the C(3) plants were relatively insensitive to CO and required a high CO to O(2) ratio of 40 to promote significant nitrate reductase activity. However, when treated with 2 millimolar 2,4-dinitrophenol, these leaves readily responded to CO even at low CO to O(2) ratios of 10 or less. On the other hand, the leaves of all C(4) plants tested, belonging to the three subgroups, were highly sensitive to CO, even at CO to O(2) ratios of 5 or less. In these leaves, the uncoupler was without any effect, probably because the mitochondria, either from mesophyll or bundle sheath cells or both, lacked tight respiratory control.

Regulation of human lymphocyte proliferation by a heterodimeric cytokine, IL-12 (cytotoxic lymphocyte maturation factor).

IL-12 is a heterodimeric cytokine that was identified on the basis of its ability to synergize with IL-2 in the induction of cytotoxic effector cells and was originally called cytotoxic lymphocyte maturation factor (CLMF). IL-12 was also found to stimulate the proliferation of PHA-activated lymphoblasts which were greater than 90% CD3+ T cells. In this report we further characterize the effects of IL-12 on lymphocyte proliferation. Studies with purified subpopulations of PHA-activated lymphoblasts and with cloned lines of human T cells indicated that IL-12 caused the proliferation of activated T cells of both the CD4+ and CD8+ subsets. This effect of IL-12 was independent of IL-2 because it was not blocked by antibodies to either IL-2 or IL-2R. The maximum proliferation induced by IL-12 was 31 to 72% of the maximum caused by IL-2; however, IL-12 was active at a lower effective concentration (EC50 = 8.5 +/- 1.3 pM) than IL-2 (EC50 = 52 +/- 8 pM). Combination of suboptimal amounts of IL-12 and IL-2 resulted in additive proliferation, up to the maximum induced by IL-2 alone. IL-12 also caused the proliferation of lymphocytes activated by culture with IL-2 for 6 to 12 days. CD56+ NK cells were among the IL-12-responsive cells in the IL-2-activated lymphocyte population. Unlike IL-2 or IL-7, IL-12 caused little or no proliferation of resting peripheral blood mononuclear cells (PBMC). In this regard, IL-12 was similar to IL-4. However, IL-12 could enhance the proliferation of resting PBMC caused by suboptimal amounts of IL-2, whereas IL-4 inhibited IL-2-induced PBMC proliferation. Thus, IL-12 is a growth factor for activated human T cells and NK cells; however, its spectrum of lymphocyte growth-promoting properties is distinct from that of IL-2, IL-4, or IL-7.

The isotype potential of B cells present in BALB/c mice chronically infected with Mesocestoides corti.

Infection of BALB/c mice with Mesocestoides corti results in a chronic infection with a pronounced splenomegaly and hypergammaglobulinemia. A prominent feature of this infection is that the vast majority of serum immunoglobulin produced is restricted to IgG1 and IgM. As much as 30-fold increases in serum IgG1 levels have been noted. To ascertain whether, as a result of infection, the resident B cell pool is committed to IgG1, B cells from infected animals were tested for their ability to produce various isotypes after stimulation. In one series of experiments, B cells from normal and infected animals were used as donor cells in the splenic fragment assay. The results show that the frequency of 2,4-dinitrophenyl-specific and phosphorylcholine-specific B cells remains unaltered in infected animals compared to controls. Importantly, the hapten-specific B cell clones induced were found to express multiple isotypes. These results demonstrate that the nonactivated B cell pool in spleens of infected mice is not committed to IgG1 and IgM production.

Effect of fetal exposure to ultrasound on the development of functional, antigen-specific B lymphocytes in fetal and neonatal BALB/c mice.

In this study, the influence of fetal exposure to ultrasound on the development of immunocompetence is addressed. Pregnant BALB/c mice were exposed to various intensities of ultrasound ranging from 0.1-3.0 W/cm2. B cells from 19 day old fetal livers or 5 and 10 day old neonatal spleens were assessed for the following: (a) differentiation into plasma cells after mitogenic stimulation with lipopolysaccharide (LPS); (b) development and frequency of 2,4-dinitrophenyl (DNP)-specific B cells, and (c) the ability to produce antibody in response to DNP. Comparison between ultrasound-exposed and sham-treated groups did not reveal any evident differences in the above tests. The results suggest that ultrasound neither hinders nor augments the development of immunocompetent B cells.

Effect of fetal exposure to ultrasound on B lymphocyte function and antibody class production.

The purpose of this study was to determine if ultrasound radiation would affect the ability of antigen-stimulated B cells and their clonal progeny to undergo the isotype switch and produce diverse antibody classes. Pregnant BALB/c mice were exposed to various intensities of ultrasound (0.1-3.0 W/cm2) at different ages of gestation. Spleens from the resulting offspring were removed five or ten days after birth. The splenocytes were analyzed for the frequency of B cells capable of responding to the hapten 2,4 dinitrophenyl (DNP) using the splenic fragment assay, a B cell cloning assay. The DNP-responsive B cell clones were then classified on the basis of isotype expression. It was found that ultrasonic radiation during gestation did not alter the frequency of B cells responding to DNP. Furthermore, ultrasound had no apparent effect on the B lymphocyte's capacity to switch to different isotypes after antigenic stimulation. Thus, the results indicate that prenatal exposure to ultrasound does not appreciably affect the genetic and cellular processes necessary for the isotype switch and antibody class production.

Effect of fetal exposure to ultrasound on B cell development in BALB/c mice.

Ultrasound is a major tool for clinical diagnosis, especially during prenatal life. Therefore, it is important that the potential bioeffects of ultrasound be determined. In this report, the effects of ultrasound on the development of B lymphocytes is studied. Pregnant BALB/c mice were exposed to intensities of ultrasound ranging from 0.1-3.0 W/cm2, receiving either a single exposure on day 11, or multiple exposures on days 14, 15, and 18 or 19 of gestation. Fetal livers were removed on day 19 of gestation whereas spleen cells were obtained from 5 and 10 day old neonates. These cell populations were analyzed for: (a) the frequency of B220+ B lineage cells; (b) the frequency of immunoglobulin-positive (Ig+) cells, and (c) the ability to proliferate in response to the B cell mitogen lipopolysaccharide (LPS). None of the tests performed revealed any substantial differences between ultrasound-exposed versus sham-treated control animals. In addition, the development of blood cell types other than B lineage cells remained unaffected by exposure to ultrasound. Therefore, exposure to ultrasound at the intensities used does not appear to hinder hemopoiesis or the normal development of B lymphocytes during fetal and early neonatal life.

Cooperative action of complement component C3 and phagocytic effector cells in innate murine resistance to Trypanosoma lewisi.

Mice display strong natural resistance to the rat-specific Trypanosoma lewisi. Intravenously injected intact T. lewisi parasites were eliminated by mice within 18 to 24 h. In comparison, "nude" T. lewisi organisms lacking the surface coat were rapidly and totally cleared from the bloodstream within 2 h postinoculation. Similarly, in vitro-cultivated trypanosomes were readily eliminated by mice with the conspicuous absence of a lag phase. Elimination of nude T. lewisi, like that of intact trypanosomes, required murine complement component C3. Splenectomy of mice did not affect their ability to eliminate T. lewisi. However, C3 depletion with cobra venom factor rendered splenectomized mice susceptible to this rat trypanosome; in these mice, T. lewisi established prolonged and frequently fatal infections. Beige mice were able to efficiently eliminate T. lewisi. But combined treatments of beige (bg/bg) and heterozygous (bg/+) mice with cobra venom factor and silica dust, or normal rat serum and silica dust, incapacitated the natural resistance of these mice to T. lewisi. Such combined treatments of beige and control mice resulted in fulminating parasitemias and death of the animals. Altogether, the results of the present studies indicate that T. lewisi elimination by mice requires the following: exposure of C3 acceptors on the surface of the parasites; activation of murine C3, probably via the alternative complement pathway; and destruction of the C3b-coated parasites by their interaction with C3b receptor-bearing, phagocytic effector cells that are abundant in the spleen and sensitive to silica dust.