Histamine release induced by dextran: the nature of the dextran receptor.

Dextrans of molecular weight 10(4) to 2 x 10(6) induced histamine release from rat peritoneal mast cells in the presence of calcium (1 mM) and phosphatidyl serine (10 micrograms/ml). Glucose and low molecular weight dextrans inhibited the histamine release induced by high molecular weight dextrans but the inhibition could not be explained in terms of a simple competitive model. Structure-activity relationships for the inhibition of dextran-induced histamine release by a number of saccharides demonstrated that substitution at the C-3, C-4, and C-6 positions of glucose were most important for activity. Inhibition of histamine release by glucose was specific for the dextran stimulus. Soluble IgE and IgG antibodies failed to interfere with histamine release induced by dextran. Phlorizin specifically inhibited the histamine release induced by dextran. Purification of mast cells on albumin or ficoll gradients produced a selective loss of response to dextran which was not due to the removal of non-mast cells but to some change in the mast cells themselves. The possible nature of the dextran receptor is discussed.

IgG receptors on the mast cells.

Antisera to the IgG subclasses, 1, 2a, 2b, and 2c, induced histamine release from mast cells obtained from the peritoneal washings of Lister hooded rats. The maximum responses obtained with anti-IgG1 and anti-IgG2a were as great as that for anti-IgE (more than 60% histamine release). Cells from unresponsive Wistar rats which did not secrete appreciable amounts of histamine in response to any of the antisera, produced on active sensitization with ovalbumin a small but significant response on challenge with anti-IgG1 and anti-IgG2b as well as with anti-IgE. Passive sensitization with rat myeloma serum of mast cells from the unresponsive rats produced a large response on challenge with anti-IgE but no release to the anti-IgG group 2 subclasses. IgE myeloma serum (1:1000) neutralized the histamine-releasing activity on anti-IgE serum (87% inhibition) and the antisera to all subclasses of IgG. When the IgE in the myeloma serum was inactivated by heating, the response to the IgG antisera remained completely inhibited except for anti-IgG2a where some reversal was observed. When purified myeloma IgE (30 microgram/ml) was used in place of whole serum, marked inhibition (86%) of the response to anti-IgE was obtained leaving the responses to the IgE subclasses unaffected (except for IgG2a, which was 65% inhibited).

Modulation of histamine secretion from Concanavalin A-activated rat mast cells by phosphatidyl serine, calcium, cAMP, pH and metabolic inhibitors.

Concanavalin A (con A) on its own released about half the histamine of peritoneal mast cells from three strains of rats. Phosphatidyl serine (PS) potentiated the release to about 80%. The secretory process had a calcium-independent component of 15--20% at pH 7.5, which increased to a maximum of 40% at pH 6.5 and nearly disappeared at pH 8.0. It was not affected by PS. In the presence of calcium and PS release was maximal and maintained at pH 7 to 8. Inhibitors of oxidative and glycolytic metabolism and dibutyryl cAMP (db cAMP) blocked secretion. The metabolic inhibitors were active whether or not PS was present, whereas db cAMP blocked much less effectively in the presence of PS. Dibutyryl cAMP reduced calcium uptake and this action was also impaired by PS. The active state induced by con A was relatively stable compared with that induced by antigen. Mast cells with a low response to anti-IgE also reponded poorly to con A on its own, but con A plus PS was a highly effective stimulus.

Failure to obtain positive MEM tests in either cell-mediated immune conditions in the guinea-pig or in human cancer.

The macrophage electrophoretic mobility test described by Caspary and Field (1971) and modified by Pritchard et al. (1973) was investigated in various models of cell-mediated immune conditions in the guinea-pig and in cancer in man. No positive results were obtained in 92 guinea-pig experiments. Only 17 of 154 experiments on 74 patients gave definite positives in experiments with human cancer and a few positive results were obtained with normal healthy subjects.

The relationship between histamine secretion and 45calcium uptake by mast cells.

1. Unstimulated mast cells from the peritoneal cavity of the rat take up (45)Ca: the initial phase of rapid uptake being complete after 1 min incubation of the cells with the isotope. Stimulation of the mast cells with an antigen-antibody reaction, dextran or concanavalin A induces an increase in the uptake of (45)Ca which is accompanied by a release of granular material: this increase in (45)Ca uptake is also complete in 1 min. The majority of the stimulated (45)Ca uptake cannot be explained in terms of binding of Ca to released granular material, or to an enlargement in either the extracellular compartment or the cell surface area.2. The magnitude of the increase in (45)Ca uptake caused by stimulating the mast cells increases when the degree of histamine secretion increases.3. The increased (45)Ca uptake induced by stimulation of the mast cells and the degree of histamine secretion are both dependent on extracellular H ion concentration. Changes of pH cause similar changes in (45)Ca uptake and secretion with maxima at pH 7.5.4. Two thirds of the (45)Ca uptake induced by an antigen-antibody reaction or by the Ca ionophore A 23187 is unaffected by inhibiting glycolysis and oxidative phosphorylation. Histamine secretion on the other hand is practically abolished by this metabolic inhibition. Thus, (45)Ca uptake proceeds in the absence of the discharge of granules.5. Dibutyryl cyclic AMP or theophylline inhibit both the increase in (45)Ca uptake and the histamine secretion caused by stimulating mast cells with an antigen-antibody reaction. Cyclic AMP, cyclic GMP and dibutyryl cyclic GMP have no effect on uptake or secretion.6. The Ca ionophore, A 23187, induces uptake of (45)Ca and histamine secretion, neither effect being inhibited by either dibutyryl cyclic AMP or theophylline.7. Phosphatidyl serine increases both (45)Ca uptake and the histamine release induced by an antigen-antibody reaction, dextran or concanavalin A.

Movement of strontium ions into mast cells and its relationship to the secretory response.

1. Non-stimulated mast cells take up (89)Sr and (45)Ca. There is a rapid phase of uptake of both labels which is complete in 1 min and the (89)Sr uptake is similar in magnitude to the (45)Ca uptake. A further slower phase of uptake occurs in the period from 1 to 30 min of incubation. The magnitude of this slower phase is about 6 times greater for (89)Sr than for (45)Ca.2. Non-stimulated and antigen-stimulated mast cells accumulate Sr which can be measured by atomic absorption spectroscopy. There is a direct relationship between Sr accumulation and histamine secretion which is independent of whether or not the cells are stimulated. 10% histamine secretion is associated with Sr accumulation of 0.25 f-mole/cell.3. The time course of (89)Sr uptake is similar to the time course of histamine secretion in non-stimulated cells.4. The uptake of (89)Sr is linearly related to the external Sr concentration in the range 0.5-16 m-mole/l. for both stimulated and non-stimulated cells.5. Ca, 10-1000 mumole/l. inhibits (89)Sr uptake in non-stimulated cells.6. Inhibition of glycolysis and oxidative phosphorylation prevents spontaneous histamine secretion in the presence of Sr without blocking the accumulation of Sr by the cells.7. Dibutyryl cyclic AMP, 10 m-mole/l. produces only 18% inhibition of (89)Sr uptake whereas histamine secretion is inhibited by 45% by the same concentration. The antigen-stimulated Sr uptake on the other hand can be completely inhibited by dibutyryl cyclic AMP, 10 m-mole/l.8. The uptakes of (89)Sr and Sr by unstimulated mast cells after 60 min incubation are dependent on extracellular H ion concentration. Both uptakes increase with increasing pH over the range pH 7-8.5.

Differential histamine release by dextran and the ionophore A23187: the actions of inhibitors.

Inhibitors of mast cell membrane activation reduced histamine release from rat mast cells induced by dextran and phosphatidyl serine but not that induced by the calcium ionophore A23187. Such inhibitors included cromoglycate, an orally-active anti-allergic agent 3-(5-tetrazolyl)thioxanthone 10,10-dioxide, dibutyryl cyclic 3':5'-AMP, theophylline and dicumarol. Inhibitors of mast cell metabolism reduced both types of release and these included oligomycin, papevevime, and the two uncouplers of oxidative phosphorylation-alpha2,4-dinitrophenol and CCCP. Inhibition of histamine release from rat isolated peritoneal mast cells by either a mixture of dextran and phosphatidyl serine or the ionophore A23187 thus allows inhibitors of mast cell membrane activation to be distinguished from those affecting cell metabolism or the later stages of the secretory process.

Inhibition by cromoglycate of histamine release from rat peritoneal mast cells induced by mixtures of dextran, phosphatidyl serine and calcium ions.

1 Dextran releases histamine from rat peritoneal mast cells in the presence but not in the absence of phosphatidyl serine (PS). With PS (10 mug/ml) present the effect of dextran was concentration-dependent in the range of 0.2-6 mg/ml.2 PS releases little histamine on its own but mixed with dextran (6 mg/ml) produces a graded effect in the range of 0.3-10 mug/ml.3 The combination of dextran and PS releases no histamine in a medium containing less than 0.1 mM calcium. As the calcium concentration is increased, release occurs and a maximum is reached at calcium 1 mM; at higher concentrations release falls off sharply.4 Histamine release by the combination of dextran (6 mg/ml), PS (10 mug/ml) and calcium (1.8 mM) is inhibited by cromoglycate in the same range (1-30 muM) that inhibits anaphylactic histamine release from rat peritoneal cells.5 Inhibition by cromoglycate occurred with all dextran concentrations tested (0.2-15 mg/ml) but high concentrations of PS (30 and 100 mug/ml) overcame the effect of cromoglycate (2 and 10 muM). Calcium concentrations above 1 mM augmented inhibition by cromoglycate 2 and 10 muM.

The action of lanthanum and manganese on anaphylactic histamine secretion.

1. In the concentration range 1 nM to 1 muM, lanthanum inhibited the calcium-dependent component of anaphylactic histamine release, but was without effect on the component which was independent of calcium.2. The inhibition of anaphylactic histamine release by lanthanum can be reversed by increasing the calcium ion concentration. The pA(2) for the lanthanum-receptor interaction was found to be 7.6.3. Lanthanum also inhibited the activation of anaphylactic histamine release by strontium ions.4. The inhibition of anaphylactic histamine release by lanthanum was reversed by eluting the lanthanum from the cells.5. In the concentration range 1 to 300 nM, lanthanum had no effect on the histamine release induced by compound 48/80 in a calcium-free medium, but the effect of calcium on the istamine release by compound 48/80 was antagonized by lanthanum in this range of concentrations.6. At concentrations of 10 muM and greater, lanthanum induced a release of histamine in the absence of antigen.7. In the concentration range 0.5 to 4.0 mM, manganese inhibited the calcium-dependent component of anaphylactic histamine release, but was without effect on the component which is independent of calcium. The inhibition of anaphylactic histamine release by manganese could be reversed by increasing the calcium ion concentration.

The interaction of calcium and strontium with phosphatidyl serine in the anaphylactic secretion of histamine.

1. Spontaneous histamine release from isolated mast cells was found to be independent of calcium in the concentration range up to 1 m-mole/l. Phosphatidyl serine did not change the effect of calcium on spontaneous release.2. Spontaneous histamine release was found to vary with the strontium ion concentration. Graded increase in the release occurred as the concentration of strontium was raised from 1 to 10 m-mole/l. Phosphatidyl serine potentiated this action of strontium; the potentiation showed a graded increase as the phosphatidyl serine concentration was raised from 1 to 100 mug/ml.3. The activation of anaphylactic histamine release by calcium was potentiated by phosphatidyl serine; the degree of potentiation showed a graded increase as the calcium concentration was raised from 0.1 to 1.0 m-mole/l.4. The activation of anaphylactic histamine release by strontium showed little, if any, potentiation by phosphatidyl serine.5. The response of the mast cells, in terms of anaphylactic histamine release, to calcium, in the presence of optimal concentrations of phosphatidyl serine, was found to be similar to that observed in the presence of strontium alone.6. These observations are discussed in terms of the concepts of affinity and efficacy of the ions at their receptor sites.