John W. Hadden

Cyclic nucleotides in mitogen-induced lymphocyte proliferation

Laboratory of Immunopharmacology, Sloan-Kettering Institute for Cancer Research, New York, N. Y. 10021 Fundamental to understanding the nature of the induction of proliferation is the elucidation of the earliest events following mitogen action and an analysis of their cause and effects. In recent years a concept has emerged that agents which induce lymphocyte proliferation do so through an action restricted to the cell surface. Greaves and Bauminger 1 showed that mitogenic plant lectins like concanavalin A (Con A) and pbytohemagglutinin (PHA) bound to Sepharose beads can induce tile proliferation of T lymphocytes without entering the cell. In addition PHA must apparently be present in optimal mito- genic concentration lot only as little as 3 h in order to trigger maximal proliferation 2. Thus, the duration and intensity of the cell surface stimulus appears to deter- mine a short-term switch-on or trigger signal process. The initial phase of lymphocyte activation following mitogen binding is characterized by nuclear activation with new RNA and protein synthesis, by blastogenesis, i.e. cellular and nuclear enlargement, and by the appearance of receptors for T-cell growth factor (TCGF or interleukin 2 (IL2). A second phase involves mitogen-dependent production of lympho- cyte activating factor (interleukin I (ILl)) by acces- sory cells, resulting in the production of IL2 by T lym- phocytes which spurs the mitogen-primed lympho- cytes to enter DNA synthesis and to undergo clonal replication as long as the IL2 is present. The process is thought then to involve a double signal - the first supplied by the mitogen for initial cellular activation which moves the cell from a restricted G~ or G~j phase of the cell cycle into a late G~ phase and the second, supplied by IL2, which moves the cell into S phase and mitosis (Fig. 1). The surface characteristics of PHA and Con A action have prompted a number of investigators to examine their role in the early events of cell activation, viewing them as hormones which exert their effects through receptors on the cell surface*. Cyclic nucleotides Parker and co-workers 4 initially observed that PHA and Con A induced early increases in the concentra- tion of lymphocyte cyclic AMP and considered this effect to be related to mitogen action. Earlier work showed that agents which increase cyclic AMP in lymphocytes inhibit the induction of proliferation by phytomitogens. This action is limited to the early phase of the triggering process. In studying the effects of PHA and Con A on the concentration of both cyclic AMP and cyclic GMP, we found that at optimal mito- genic concentration, both mitogens increased cyclic GMP 5 but that neither mitogen influenced the con- centration of cyclic AMP. The ability of plant lectins to increase the cellular concentration of cyclic AMP in Iymphocytes has remained controversial. The disputes appear to rest on differences in incubation conditions, mitogen con- centrations, and nucleotide assay methods. While most investigators have observed that optimal mito- genie concentrations of PHA and Con A do not increase the concentration of cyclic AMP in the lymphocyte a number have found that at high doses cyclic AMP concentrations are raised while the mito- genic effect is lost (high dose inhibition). It is at these high concentrations that one clearly observes the agglutinating activity and the induction of patch and cap formation by these agents. Edelman and co- workers 6 showed that mitogenic succinylated subunits of Con A do not produce patch and cap formation and also do not show high dose inhibition characteristic of *These studies were reviewed in 1978 and the reader is referred to Ref. 3 for more complete referencing of the pre-1978 studies.

Cyclic GMP and lymphocyte proliferation: effects on DNA-dependent RNA polymerase I and II activities.

Abstract Cyclic GMP (guanosine 3′:5′-cyclic monophosphate) accumulation and Ca2+ influx have been correlated with early nuclear events associated with increases in RNA polymerase I activity and decreases in RNA polymerase II activity in phytohemagglutinin-stimulated lymphocytes. In the present study we demonstrate that cyclic GMP in the presence of Ca2+ stimulates RNA polymerase I activity in lymphocyte nuclei isolated from both non-stimulated and phytohemagglutinin-stimulated lymphocytes. In addition, cyclic GMP in the presence of Ca2+ decreases RNA polymerase II activity in both non-stimulated and phytohemagglutinin-stimulated lymphocyte nuclei. These observations suggest that cyclic GMP and Ca2+ may represent components of a plasma membrane-to-nucleus “mitogen signal sequence”.

Glucocorticoid modulation of lymphokine-induced macrophage proliferation

Abstract The ability of glucocorticoids to modify lymphokine-induced macrophage proliferation, an in vitro correlate of cellular immunity in the guinea pig, was investigated. Lymphocyte production of macrophage mitogenic factor (MMF) was decreased in the presence of physiological concentrations of glucocorticoids. Inhibition was concentration dependent (IC 50 of triamcinolone acetonide (TA): 2 × 10 −9 M ), glucocorticoid specific, and reversed by cortexolone. In contrast, pharmacological concentrations of glucocorticoids were necessary to inhibit macrophage proliferation induced by suboptimal dilutions of MMF. This inhibition was concentration dependent (IC 50 of TA: 4 × 10 −7 M ), glucocorticoid specific, and reversed by cortexolone. At supraoptimal dilutions of MMF, glucocorticoids caused a twofold potentiation of MMF-induced macrophage proliferation. Potentiation was concentration dependent (EC 50 of TA: 3 × 10 −8 M ), glucocorticoid specific, reversed by glucocorticoid antagonists, and occurred in the presence of indomethacin. Thus, glucocorticoids regulate both the initiation and effector phases of this in vitro model of delayed hypersensitivity. However, the results indicate that the major mechanism of glucocorticoid-mediated anti-inflammatory action occurs at the level of the MMF-producing lymphocyte rather than at the effector macrophage, as MMF-induced proliferation is likely controlled by opposing glucocorticoid-sensitive mechanisms.

Immunoenhancing activity of NPT 15392: A potential immune response modifier☆

NPT 15392, a new immunomodulating compound related to inosine in structure and isoprinosine in action, enhances T-cell dependent immune responses. Antibody responses to sheep red blood cells are augmented two to threefold in mice receiving NPT 15392 while T-cell independent antibody responses to TNP-LPS are unaffected. NPT 15392 does not enhance or alter the number of clonable B cells. This drug also increases cytotoxic T-lymphocyte responses to allogeneic tumor cells but does not alter the number of cytotoxic precursor cells. Immature hematopoietic cell classes (clonable progenitor cells) were also monitored and found not to be influenced by NPT 15392.

Immunomodulatory effects of cyclomunine in vitro

Abstract Cyclomunine, a cyclic polypeptide extracted from Fusarium equiseti , is thought to be an immuno-modulator. Low concentration of cyclomunine (1 ng/ml) decreases lymphocyte nuclear refringence and potentiates the response to suboptimal concentrations of concanavalin A (Con A) in the absence of an effect on proliferation. At this concentration, cyclomunine potentiates Con A-induced suppressor cell function. Cyclomunine at concentrations of 1–10 μg/ml increases lymphocyte nuclear refringence and inhibits the proliferative responses of human lymphocytes to mitogens and of guinea-pig macrophages to a lymphokine. The inhibitory effect of cyclomunine is observed on the proliferation of mitogen-induced lymphoblasts and of human leukemic cells at a 10-fold lower concentration than of macrophages. Cyclomunines cytotoxic index correlates with the growth inhibitory index for leukemic cells and mitogen-induced lymphoblasts. Resting lymphocytes are more resistant to the cytotoxic effects. The data indicate that cyclomunine at low concentrations may have selective effects to modulate lymphocyte helper or suppressor function, while at higher concentrations is toxic to lymphocytes. Collectively, the results indicate that cyclomunine is a new immunoregulatory compound with potent biological activity.

Lymphokine-mediated fusion and migration inhibition of alveolar macrophages

Abstract Guinea pigs were shown to produce a lymphokine termed macrophage fusion factor (MFF) which mediated the fusion of 70–80% of guinea pig or rabbit alveolar macrophages, but not guinea pig peritoneal macrophages. In the conventional migration inhibitory factor (MIF) assay, guinea pig aveolar macrophages were inhibited in their migration and large numbers of giant cells were present. There appeared to be a correlation between the titer of MFF and migration inhibition of alveolar macrophages but not with MIF titer as expressed on the peritoneal macrophage. Guinea pig MFF production was erratic and its absence from lymphokine supernatant fluids correlated with an absence of migration inhibitory activity for the alveolar macrophage. Guinea pig MIF production was more constant and high titers were invariably present. Rabbit crude lymphokine supernatant fluids containing MFF also inhibited the migration of their alveolar macrophages when measured at 24 and 48 hr during the MIF assay. Extensive numbers of giant cells were observed in the cell fan whenever migration inhibition was present. α- l -Fucose, which is known to block the receptor sites of MIF, failed to block giant cell formation in either the MFF or the MIF assay and also failed to block migration inhibition of the alveolar macrophages. The results suggest that lymphokines other than MIF can inhibit the migration of alveolar macrophages in the standard MIF assay and that the lymphokine responsible for migration inhibition and fusion of alveolar macrophages is the same lymphokine, MFF.

Cyclomunine: a potent inhibitor of rat and canine lymphocytes.

In the present study we examined the effect of a recently isolated hexacyclodepsipeptide, Cyclomunine (Servier, France), on certain parameters which assess cellular and humoral immunity in the rat and dog. Our data establish Cyclomunine as a potent in vitro inhibitor of 3H-thymidine incorporation by lymphocytes. The concentrations of Cyclomunine required to abrogate mitogen or allogeneic lymphocyte stimulated 3H-thymidine incorporation was greater in the dog than in the rat, 15 micrograms/ml versus 10 micrograms/ml, respectively, to achieve greater than 99% inhibition. Little cytotoxicity was noted at concentrations below 25 micrograms/ml. Oral administration of Cyclomunine to rats (50 mg/kg dissolved in corn oil) did not significantly alter white blood cell count, lymphocyte-polymorphonuclear cell differential, body weight, or histological architecture of the liver, spleen, lymph node or kidney. Our results show that in vitro, Cyclomunine is highly effective in suppressing lymphocyte proliferation. In vivo, Cyclomunine appears to have little myelosuppressive effect a property shared with Cyclosporin A. Cyclomunine may hold promise as a clinically useful immunosuppressive agent.