Mechanisms of action of “lymphocyte-activating factor” (LAF): IV. Differential stimulation of T lymphocytes by induced macrophage enzymes (catheptic carboxypeptidase B and serine proteases)

PHA and concanavalin A are unable to induce DNA synthesis in a substantial proportion of highly purified rat lymph node T cells in serum-free culture. Addition of dialyzed macrophage supernatant (LAF or TAF) to these mitogen-stimulated cells, even as a 12-hr pulse starting at 14 hr, gives a stronly potentiated response, while LAF (TAF) alone is nonmitogenic. Thus mitogen provides a first signal and LAF (TAF) a second signal in time. General protease inhibitors (trasylol, soybean trypsin inhibitor, and especially ?-aminocaproic acid) markedly inhibit both the low response to mitogen alone and the LAF effect, while certain more narrowly specific inhibitors (phenylmethylsulfonyl fluoride, tosyl-lysl-chloromethyl ketone, iodoacetate) do not. This finding suggests that the LAF effect involves protease action. The LAF effect has been shown to parallel the Carboxypeptidase B content of various LAF preparations and can be mimicked by commercial pancreatic Carboxypeptidase B. A lesser LAF effect is also induced by such serine proteases as trypsin, chymotrypsin, and plasmin, none being mitogenic alone. Thus LAF (TAF) may represent the combined action of several enzymes. These findings are contrasted with the demonstrated ability of many serine proteases to stimulate DNA synthesis, i.e., to serve as first signal, in B cells. LAF (TAF) acts synergistically with commercial carboxypeptidase B. This implies that they may act on different target sites in the cell membrane and that triggering of an adequate number of sites is required for an effective second signal. LAF (TAF) interaction with T cells is not cell cycle specific since activity is absorbed by unstimulated cells and by cells at various times after mitogen exposure.     

Leukocyte migration inhibitory factor: a serine esterase released by stimulated human lymphocytes. Kinetic analysis and inhibition by cyclic GMP.

Lymphocytes, stimulated with concanavalin A, release small amounts of non-immunoglobulin, highly reactive proteins called lymphokines. One of these, a serine esterase, termed leukocyte migration inhibitory factor according to its function in vitro, is found in supernatants of stimulated human lymphocytes at concentrations less than 1 ng/ml. The esterase was purified in good yield and its esterolytic activity was measured by a sensitive radioenzymic assay. The kinetics of the esterolytic activity were studied and the effect of various nucleotides examined. Competitive inhibition of esterolysis was seen with cyclic GMP at concentrations down to 10(-7) M, and with 2',3'-cyclic CMP at a concentration of 10(-3) M. A role of this esterase, not only as a mediator acting upon polymorphonuclear leukocytes, but also as an intracellular regulator of lymphocyte activation, is discussed.     

Characterization of lymphocyte-activating factor (LAF) produced by a macrophage cell line, P388D1. II. Biochemical characterization of LAF induced by activated T cells and LPS.

Unstimulated P388D1 cells, as well as P388D1 cells stimulated with PHA-activated guinea pig T lymphocytes or LPS, produced a lymphocyte activating factor (LAF). In order to have a chemical basis for comparing this LAF with the LAF produced by normal macrophages, we have analyzed several biochemical characteristics of the P388D1-derived LAF. Sephadex G-75 chromatography of concentrated LAF-containing supernatants from cultures of unstimulated and T cell stimulated P388D1 cells demonstrated that the cell line LAF had a m.w. of approximately 16,000. On DEAE cellulose, the T cell-induced LAF fractionated into at least three major peaks and one minor peak. By using hydroxylapatite chromatography, two of the major peaks of LAF activity were separated from residual contaminating Lowry positive material. LPS-stimulated P388D1 also produced LAF with a m.w. of 16,000. However, the LPS-induced LAF appeared to lack one of the DEAE peaks of LAF activity observed with the T cell-derived LAF. In contrast to LPS, T cells may induce the synthesis and/or release of an additional LAF component or enzymatically modify one or more of the LAF species that are produced in response to both stimulants. Based on the results of chemical characterization studies, the P388D1-derived LAF appears to be similar in size and charge to the lymphocyte activating factor produced by normal macrophages.     

Absence of an effect of the lithium-induced increase in cyclic GMP on the cyclic GMP-stimulated phosphodiesterase (PDE II). Evidence for cyclic AMP-specific hydrolysis

Chronic treatment of rats with LiCl is known to induce a decrease in cAMP, while this decrease has also been found to occur together with both a simultaneous increase in total cortical phosphodiesterase (PDE; EC 3.1.4.17) activity and a concomitant increase in cGMP. These studies have implicated an involvement of PDE in lithium (Li⁺) action and it has been suggested that cGMP and the cGMP-stimulated PDE may be instrumental in the observed effects of Li⁺ on cAMP. In this study, three isozymes of PDE were isolated and identified from rat cortex and their activity determined, together with simultaneous measurement of cAMP and cGMP, after chronic treatment with oral LiCl (0.35% m/m). Li⁺ treatment exerted profound effects on cyclic nucleotides in the cortex, inducing significant suppression of cAMP while increasing cGMP levels. However, the ion only induced a slight but insignificant increase in the activities of the three PDE isozymes. To confirm these observations, methylparaben (MPB), a drug demonstrating both an ability to induce a selective stimulation of cAMP-specific PDE and also to lower intracellular levels of cGMP, was co-administration orally (0.4% m/m) with Li⁺ over the same period. This combination emphasized certain actions of Li⁺ not noted with Li⁺ alone. MPB inhibited the Li⁺-induced increase in cGMP, yet did not prevent the ion from decreasing cAMP. However, the combination of Li⁺ and MPB engendered a synergistic 100% increase in the activity of the membrane-bound, cAMP-specific PDE, PDE IV. This combination also produced a significant suppression of cAMP, while no reduction in cGMP was observed. The data is indicative that Li⁺-induced suppression of cAMP does not appear to be related to an effect on the cGMP-dependent PDE II, and that the increases in cGMP and PDE induced by Li⁺ observed previously and in the present study are two unrelated events. Instead, the synergistic response of Li⁺ plus MPB on PDE IV, and the associated reduction of cAMP, indicate that Li⁺ may promote selective cAMP hydrolysis via an effect on membrane-bound forms of PDE. This effect of Li⁺ on PDE IV, as well as the reciprocal effects on cyclic nucleotide balance, may have important implications in explaining the antipsychotic actions of the ion.     

Regulatory substances produced by lymphocytes. III. Evidence that lymphotoxin and proliferation inhibitory factor are identical and different from the inhibitor of DNA synthesis.

L cell mutant lines which were considerably more resistant to rat lymphotoxin (LT) than the original cell line were obtained by periodic additions of LT, partially purified from sensitized lymph node cell culture supernatants by DEAE-cellulose chromatography and Sephadex gel filtration. Addition of actinomycin D to cultures of these mutant cells abrogated resistance to LT, suqgesting that resistance was not due to a loss of LT receptors but probably to increased activity of a repair mechanisms. These mutant lines were also more resistant to the proliferation inhibitory effect of LT in low concentration and to that of diluted culture supernatants of lymph node cells stimulated with antigen (ovalbumin) than the original cell line, but they remained as sensitive to inhibitor of DNA synthesis (IDS) as the original line. The mutant lines also remained fully sensitive to both complement-dependent lysis by antibody and antibody-dependent cellular cytotoxicity, but showed increased resistance to the killing effect of rat lymph node cells sensitized with orginal L cells in vivo. These findings suggest that the lymphotoxic substance partially purified and characterized in this and in the previous paper may be an important mediator of T cell-mediated cytotoxicity.     

Evidence that the adaptive response of human lymphocytes to ionizing radiation acts on lethal damage in nonaberrant cells

In a previous study we found that a cytogenetic adaptive response could lead to increases in survival if there was a sufficient increase in nonaberrant cells (Shadley and Dai, 1992). Since the high challenge doses used produced mainly multiply aberrant cells, we suggested using challenge doses that gave mainly singly aberrant cells in order to improve detection of a survival adaptive response. To test this, human lymphocytes from 6 donors were exposed in the first G₁ phase to 5 cGy of X-rays, followed by 100 cGy 6 h later. Nearly all of the aberrant cells bore only one chromosome aberration with this challenge dose, and in agreement with our proposal, survival adaptive responses were seen in 4 of 6 donors. A near 1:1 relationship between the % nonaberrant cells and % survival was found with 100 cGy, suggesting that the lymphocyte populations scored in the survival and aberration assays were representative of each other. However, the increase in nonaberrant cells was not sufficient to account for the increase in survival. Thus, a large fraction of the increase in survival was due to a decrease in lethal damage in cytologically nonaberrant cells. Such damage could range sub-microscopic lesions, to larger alterations not visible in Giemsa-stained cells. In conjunction with adaptive response studies of others, these results intimate that the adaptive response affects damage at different levels of chromosomal hierarchy (i.e. from the chromosome to DNA). The process(es) responsible for the effects observed in this study may act on lethal, rather than mutagenic lesions.     

Relationship between production and release of lymphocyte-activating factor (interleukin 1) by murine macrophages. 1. Effects of various agents

The relationship between the production and release of lymphocyte-activating factor (LAF, or interleukin 1) by cultured murine peritoneal macrophages (Mφ) was investigated. Unstimulated Mφ produce high levels of intracellular LAF within a few hours after culturing, but release little of this activity into their culture medium. Addition of various agents was found to increase significantly the production and release of LAF, with three different patterns: (a) Both intracellular and extracellular LAF activities were increased in response to latex beads, (b) A marked increase of intracellular LAF, with just a minimal elevation of extracellular activity, was stimulated by LPS. (c) Sharp increases in LAF release, with small increments of the intracellular activity, were induced by silica and glucocerebroside (GL₁). Silica and GL₁ damaged the cultured Mφ, as indicated by the increased release of lactate dehydrogenase. It is significant, therefore, that silica and GL₁ increased both intracellular and extracellular LAF levels, suggesting that damage of Mφ may stimulate total LAF production. A combination of LPS with silica or GL₁ acted synergistically on Mφ to release very high levels of LAF, which far exceeded those released by the individual agents. The agents were also tested on Mφ which were precultured, to deplete their LAF content. Latex, LPS, or silica increased LAF production and release by precultured Mφ, but the levels were lower than those obtained with freshly cultured Mφ. The results of this study thus show that the level of LAF release does not necessarily reflect the level of total LAF production by cultured Mφ and suggest that injurious agents may promote LAF production.     

Mechanisms of action of histidinol dehydrogenase and UDP-Glc dehydrogenase. Evidence that the half-reactions proceed on separate subunits.

Histidinol dehydrogenase and UDP-Glc dehydrogenase catalyze 4-electron dehydrogenations that convert primary alcohol groups to the corresponding acids. Both reactions proceed in two distinct steps involving the oxidation of the primary alcohol to a bound form of the intermediate aldehyde, followed by oxidation of this to the corresponding acid. The enzymes have subunit structure, the former is made up of two subunits and the latter of six (beef liver enzyme). Evidence is presented that the two half-reactions proceed independently of the overall reaction. Histidinol dehydrogenase preparations that approach total dissociation into subunits also approach total inhibition of the overall reaction, while the second half reaction is completely unaffected and 50% of the first half-reaction survives. Further, the fraction of overall activity surviving in partially dissociated preparations follows the weight fraction of residual dimer. UDP-Glc dehydrogenase behaves in an analogous fashion. These data are interpreted on the basis that both enzymes function by carrying out first oxidation step at a site on one subunit and then pass the intermediate to a vicinal site on the adjacent subunit, where the reaction is completed.     

Changes in cyclic AMP and cyclic GMP concentrations during the action of 5-hydroxytryptamine on an insect salivary gland.

Salivary glands from adult blowflies (Calliphora erythrocephala Meigen) were studied in vitro. The time course of changes in cyclic AMP content of the glands was followed at different concentration of 5-hydroxytryptamine. There was an immediate biphasic rise and fall in cyclic AMP content, following by a slower rise and subsequent gradual decline. The initial rise preceded the onset of fluid secretion by the glands. Rises in cyclic AMP content were inhibited by compound RMI 12330 A (an adenylate cyclase inhibitor) and were halted after about 15-20s if the glands were deprived of Ca2+. Theophylline (a phosphodiesterase inhibitor) abolished the decline phase of the fast response, Losses of cyclic AMP from the glands either to the bathing medium or to the saliva were small and could not account for the rapid fall found. Evidence is presented that cyclic GMP is not involved in the process of initiating secretion in the blowfly salivary gland.     

Studies on the role of lymphocyte-activating factor (Interleukin 1) in antigen-induced lymph node lymphocyte proliferation

The in vitro proliferation of primed lymph node lymphocytes (LNL) in response to the soluble antigen ovalbumin (OVA) was dependent upon the presence of adherent cells. Restoration of OVA-induced LNL proliferation could be achieved by addition of highly purified lymphocyte-activating factor (LAF; Interleukin 1, IL 1): LAF (IL 1) did not stimulate LNL proliferation in the absence of the priming antigen or T lymphocytes. Furthermore, treatment of the LNL with antimacrophage serum completely blocked the ability of the LNL to respond to OVA and LAF (IL 1), suggesting that the residual macrophages in the LNL population were necessary to provide an additional function or signal, possibly antigen presentation, in conjunction with LAF (IL 1). These data therefore support the two signal hypothesis of macrophage-mediated lymphocyte activation and demonstrate the ability of LAF (IL 1) to provide one of these signals.     

Immunofluorescence of histone H1 in stimulated lymphocytes measured by flow cytophotometry.

The modification of histones or their redistribution during the transition from actively transcribing chromatin to the heterochromatic chromosomes seems to play a major in regulation of gene expression. The purpose of this study was to monitor the change in immunofluorescence of histone HI during phytohemagglutinin stimulation in peripheral lymphocytes. The histone antigens were prepared from pig thymus, proven to be pure by gel electrophoresis and repeatedly injected as RNA-complexes into rabbits. The antihistone HI antiserum titer was 1:4000, and there was no cross-reactivity with other histone fractions as shown by microcomplement fixation tests. Affinity chromatography purified antibody after being labeled with fluorescein isothiocyanate was able to differentially stain HeLa cells as controls and those, where histone HI had been extracted by perchloric acid treatment. The measurements were done on a Los Alamos Scientific Laboratories-flow cytophotometer cell sorter. Staining peripheral lymphocytes resulted in a bimodal distribution. The increase in number of cells with high fluorescence intensity had its maximum about 20 hr before the maximum proliferative activity of the lymphocytes as measured by number of cells in S phase with the DNA-stain mithramycin.     

Steroidogenic effect of atrial natriuretic factor in isolated mouse Leydig cells is mediated by cyclic GMP.

The effects of different atrial natriuretic peptides on cyclic GMP formation and steroidogenesis have been studied in Percoll-purified mouse Leydig cells. Rat atrial peptides rANP (rat atrial natriuretic peptide), rAP-I (rat atriopeptin I) and rAP-II (rat atriopeptin II), in the presence of a phosphodiesterase inhibitor, stimulated cyclic GMP formation in a concentration-dependent manner. In the presence of saturating concentrations of the peptides, a 400-600 fold stimulation of cyclic GMP accumulation was observed. Among the peptides, rAP-II appeared to be the most potent. ED50 values (concentration causing half-maximal effect) for rAP-II, rANP and rAP-I were 1 X 10(-9) M, 2 X 10(-9) M and 2 X 10(-8) M respectively. A parallel stimulation of cyclic GMP formation and testosterone production by the cells was observed after incubation of the cells with various concentrations of rAP-II. In the presence of a saturating concentration of rAP-II (2 X 10(-8) M), maximum stimulation of intracellular cyclic GMP content was obtained within 5 min of incubation. Testosterone production by mouse Leydig cells could be stimulated by 8-bromo cyclic GMP in a concentration-related manner. At a 10 mM concentration of the cyclic nucleotide, steroidogenesis was stimulated to a similar extent as that obtained with a saturating concentration of human chorionic gonadotrophin (5 ng/ml). On the basis of these results we conclude that cyclic GMP acts as a second messenger in atrial-peptide-stimulated steroidogenesis in mouse Leydig cells. The steroidogenic effect of atrial peptides appears to be species-specific, since none of these peptides stimulated testosterone production by purified Leydig cells of rats, though in these cells a 40-60-fold stimulation of cyclic GMP formation in response to each of the three peptides was observed. However, 8-bromo cyclic GMP could stimulate testosterone production in rat Leydig cells. Therefore we conclude that the lack of steroidogenic response in rat Leydig cells to atrial-natriuretic-factor-stimulation results from an insufficient formation of cyclic GMP in these cells. This species difference would appear to result from a lower guanylate cyclase activity in rat Leydig cells.     

Direct action of melatonin on testosterone and cyclic GMP production using rat testis tissue in vitro.

When $\text{Escherichia}\text{coli}$ is growing in steady state with doubling times of 40 minutes or 20 hours, during shift up conditions, or under conditions of relaxed control of RNA synthesis, all rRNA cistrons are transcribed equally.     

Characterization of the spontaneous murine B cell leukemia (BCL1). III. Evidence for monoclonality by using an anti-idiotype antibody.

We have raised an anti-idiotypic antibody against the cell surface IgM of the murine BCL1 tumor cells. This antiserum reacts exclusively with the IgM expressed on the tumor cells and detects a unique population of cells in the spleen and blood of the tumor-bearing mice. When these cells are stimulated in vitro with LPS, they secrete an IgM bearing the same idiotype as the cell surface Ig. These results are discussed in terms of a model for the immunotherapy of a chronic lymphocytic leukemia-like syndrome in mice.