Studies on the inhibitory effects of analogues of dapsone on neutrophil function in-vitro.

We have compared twelve sulphone analogues of dapsone in terms of inhibition both of zymosan-mediated human neutrophil respiratory burst and inhibition of interleukin-1-stimulated neutrophil adhesion to transformed human umbilical vein endothelial cells. Overall, there was a good correlation between the respective rank orders of compound potency in the two test systems. The most effective compounds in terms of respiratory burst and adherence inhibition were the 2-nitro-4-amino-, 2-hydroxy-4-aminopropyl-, and 2-methoxy-4-aminoethyl- derivatives. In general, potency was inversely associated with lipophilicity; compounds with bulky side-chains, e.g. the 2-methyl-4-aminopentyl, 2-methyl-4-aminohexyl and the 2-hydroxymethyl-4-aminoethyl derivatives, were less potent. A 2-hydroxy-4-amino- derivative was the exception, however, with low lipophilicity and relatively low potency. All of the compounds tested showed comparable or greater inhibition in both the neutrophil-mediated assays compared with dapsone. Some of the compounds might, because of their good tissue penetration and lower toxicity than dapsone, have the potential to undergo further development.

Preliminary evaluation of the toxicity and efficacy of novel 2,4-diamino-5-benzylpyrimidine-sulphone derivatives using rat and human tissues in vitro.

Four novel combined dapsone and trimethoprim analogues, K-120, K-150, K-138 and DRS-506, have been compared with dapsone in their methaemoglobin forming abilities as well as their anti-inflammatory properties using rat and human tissues in vitro. All four compounds formed consistently less methaemoglobin compared with dapsone in both the rat and human microsomes. Using human microsomes from five livers, K-120 was significantly less toxic than the other analogues in three of the five livers (P < 0.01). DRS-506 and K-138 both inhibited the human neutrophil respiratory burst to a significantly greater degree compared with dapsone at 0.5 mM (P < 0.01), while K-120 and K-150 showed no significant effect at 0.5 mM. At 1 mM, DRS-506, K-120 and K-138 were more potent than dapsone (P < 0.01), although K-150 appeared to increase the neutrophil activation. All four analogues caused a significant reduction in neutrophil adhesion to human umbilical vein cells at 0.1 mM. In view of its efficacy and low toxicity, K-120 shows considerable promise for future clinical evaluation.

Parathyroid hormone stimulation of mitosis in rat thymic lymphocytes is independent of cyclic AMP.

The in vitro mitotic response of rat thymic lymphocytes to hPTH(1-34), hPTH (1-38), and 8,18 Nle hPTH(1-34) exhibits a dependency upon extracellular calcium. Removal of extracellular calcium or the addition of Verapamil (5 micrograms/ml) or trifluoroperazine (10 microM) abrogated the mitotic response. Mitogenic concentrations of 8,18 Nle hPTH(1-34) increased calcium 45 uptake from 4.49 +/- 0.25 to 8.23 +/- 0.75 pMol/10(6) cells/min. The intracellular calcium concentration, measured by Quin 2 fluorescence, also increased after addition of 8,18 Nle hPTH(1-34). Parathyroid hormone-induced activation could not be demonstrated in an otherwise responsive thymocyte membrane adenylate cyclase. In intact cells mitogenic levels of 8,18 Nle hPTH(1-34) decreased intracellular cyclic AMP content. This response was blocked by both 3-isobutyl 1-methyl xanthine and trifluoroperazine, and may indicate activation of calcium-dependent phosphodiesterase. We conclude that PTH stimulates thymic lymphocyte proliferation independently of cyclic AMP, and that changes in cellular calcium homeostasis are intimately involved in the action of PTH. In all of the assays employed, the hitherto antagonistic analogue 8,18 Nle 34 Tyr bPTH(3-34)amide proved to be an agonist. We postulate that the receptor utilized for this PTH action may not exhibit classical PTH structure-activity specificities.

In vivo administration of interleukin 2 stimulates mitosis in thymus and bone marrow.

We have used short-term, high-density cultures to demonstrate that interleukin 2 (IL 2) in picomolar amounts causes entry of approximately 2% of thymocytes from 3-month-old rats into mitosis. Newborn and fetal animals show a higher response reflecting a greater proportion of cells which have been shown to express IL 2 receptors at this age. In vivo administration of nanogrammes of IL 2 or injection of rats with syngeneic spleen cells which had been stimulated in vitro with concanavalin A to release IL 2 were also shown to increase the proliferation of both thymus and bone marrow cells. This suggests that IL 2, in amounts which could be produced by peripheral lymphoid tissue during immune responses, could act to increase the turnover of lymphocytes in bone marrow and thymus.

Interleukin 2 stimulates T cell proliferation using a calcium flux.

A preparation enriched in rat interleukin 2 caused enhanced DNA synthesis in an interleukin 2-dependent mouse cytotoxic T cell line, in lectin transformed mouse splenocytes and in rat thymocytes. The enhanced proliferation due to interleukin 2 could be abrogated by chelating calcium from the culture medium or blocking calcium entry into the cells. Compounds which interfere with the function of calmodulin also inhibited proliferation. The addition of interleukin 2 to IL-2 dependent cells caused an increase in the intracellular concentration of calcium ions, as measured using Quin 2. The requirement for IL-2 by blasts and thymocytes could be replaced by calcium ionophore. The results implicate a calcium flux as an essential component of the action of interleukin 2 on its target cells.

The promotion of mitosis in cultured thymic lymphocytes by acetylcholine and catecholamines.

Thymic lymphoblasts possess beta-adrenergic, dopaminergic and nicotinic receptors. When activated by high concentrations of adrenaline, isoprenaline, dopamine and acetylcholine, magnesium-dependent events are initiated, which culminate in mitosis. These events can be blocked by testosterone. The cells also possess muscarinic and alpha-adrenergic receptors which respond to low concentrations of acetylcholine, carbamylcholine and noradrenaline. In these cases calcium-dependent, oestradiol-blockable mechanisms are triggered which eventually lead to cell division.

Some effects of parathyroidectomy on cell-mediated immune responses in the rat.

Xenografts of mouse tail skin to the rib cages of normal and sham-parathyroidectomized rats caused an increase in plasma calcium concentration and concomitant increase in bone marrow mitosis. Neither was elicited in aparathyroid rats and graft survival was prolonged in these animals. No hypercalcaemic episode was associated with the delayed hypersensitivity response induced by painting rat ears with oxazolone. Compared with the response in sham-parathyroidectomized rats, that in parathyroidectomized rats was enhanced although both responses were less than that in normal rats. Parathyroidectomy of parental donors did not affect the ability of their splenic lymphocytes to mount a graft-versus-host response in F1 hybrid recipients. When sham-operated and aparathyroid parents were sensitized with F1 hybrid lymphocytes no differences were observed in a subsequent graft-versus-host response in F1 recipients. However, when aparathyroid F1 recipients were employed a marked reduction in the graft-versus-host reaction was observed. Thus the clonal expansion of cells with specific reactivity to certain antigens in secondary lymphoid tissue, which is driven by those same specific antigens, is not affected or only moderately affected by the parathyroid status of the animal. However, the more general increase in lymphocyte numbers promoted by non-specific mitogenic lymphokines is markedly impaired in the hypocalcaemic parathyroidectomized rat. Furthermore, the parathyroid gland is essential for the development of a hypercalcaemic episode which follows antigenic challenge and causes cell proliferation in primary lymphoid tissues. This surge of mitosis could serve to replenish the depleted pools of virgin T and B lymphocytes in the secondary lymphoid tissue which occur as a result of their response to antigens.

Sodium and ouabain induce proliferation of rat thymic lymphocytes via calcium- and magnesium- dependent reactions.

When the concentrations of either calcium or of magnesium in the culture medium were increased from the normal 0.6 and 1.0 mM to 1.8 and 2.5 mM respectively mitotic activity of rat thymic lymphocytes increased. Very high (10(-4)M) ouabain concentrations abolished these mitogenic actions whilst lower (10(-7) and 10(-11)M) concentrations had no effect. However in the normal medium these lower concentrations of ouabain were themselves mitogenic. The stimulatory effect of 10(-7)M ouabain was calcium-dependent and oestradiol-blockable and that of 10(-11)M magnesium-dependent and testosterone-blockable. A 10 mM increment in extracellular sodium concentration also stimulated mitosis in these cells in a calcium-dependent manner whilst a 20 mM increment required the presence of magnesium to exert its mitogenic effect. However, when similar osmotic increments were provided by potassium and lithium salts, or sucrose no mitotic stimulation was provoked. Subtle interactions between sodium and the divalent cations are clearly involved in events which lead to mitosis and the steroids oestradiol and testosterone can somehow block these effects.

Changes in plasma levels of calcium and in bone marrow mitosis after antigenic challenge in rats and mice.

When rats or mice were immunized with sheep red blood cells, bacterial lipopolysaccharides or bovine serum albumin, a proliferative response could be detected in the bone marrow and spleen. This response was associated with a hypercalcaemic phase. Parathyroidectomy, which resulted in a protracted hypocalcaemia, prevented the development of an increase in levels of plasma calcium. This operation also prevented the rise in bone marrow proliferations following antigenic challenge, but did not ablate the normal proliferative response to antigen by cells in the spleen. Antibody production and numbers of antibody-forming cells were not significantly reduced by parathyroidectomy. These results suggest that there is a pool of antigen-insensitive cells in the bone marrow which are stimulated after antigenic challenge. It is postulated that these events were mediated by the development of a parathyroid-dependent hypercalcaemia which stimulates the cells non-specifically. These events may form part of a cellular homeostasis, replacing cells in peripheral lymphoid tissues.

Role of vasopressin in the mitotic response of rat bone marrow cells to haemorrhage.

Two days after a severe haemorrhage plasma calcium concentrations and bone marrow mitotic activity in rats were significantly increased and so remained for a further 5-6 days until the haematocrit had returned to normal. The first 48 h after bleeding were characterized by hypocalcaemia. During this phase two significant peaks in mitotic activity were observed at 4 and 18 h after haemorrhage. The mitotic surge 4 h after bleeding was still present in adrenalectomized and parathyroidectomized animals but in rats which were either hypophysectomized or had congenital diabetes insipidus this mitotic response was absent. Vasopressin was shown to stimulate bone marrow mitotic activity both in vivo and in vitro whereas angiotensin, aldosterone and erythropoietin had no rapid, direct mitogenic action on these cells. This novel hypophysial-bone marrow system suggests that vasopressin may assist in post-haemorrhagic recovery in blood cell numbers in the circulation.