IMMUNOLOGICAL INDUCTION OF INCREASED VASCULAR PERMEABILITY : II. TWO MECHANISMS OF HISTAMINE RELEASE FROM RABBIT PLATELETS INVOLVING COMPLEMENT

Two mechanisms have been described whereby rabbit platelets in vitro may be induced to release their contained histamine by the reaction of antigen and antibody. Both processes require the participation of the complement system. In the first, the adherence of platelets to particulate antigens such as zymosan or erythrocytes which have fixed complement through the third component was followed by histamine release. Plasma lacking C6 activity was fully active in this system. In the second mechanism, the reaction of soluble antigen with antibody in the presence of plasma also caused release of histamine from platelets and platelet clumping was observed. The release process, which appeared to follow the adherence of platelets to the immune complex, required the action of C6 and perhaps the later-acting components. No evidence could be obtained that a soluble factor was produced which caused the release. Instead, an inhibitor of the release process was detected after incubation of antigen, antibody, and plasma. Antibody preparations capable of giving complement-dependent PCA reactions in rabbits were also shown to induce the release of histamine from platelets in vitro.     

Cultured endothelial cells synthesize both platelet-activating factor and prostacyclin in response to histamine, bradykinin, and adenosine triphosphate.

Cultured human endothelial cells synthesize prostacyclin (PGI2), a potent inhibitor of platelet function, when stimulated with histamine, bradykinin, or ATP. Paradoxically, we report that these agonists also induced the rapid and sustained synthesis of platelet-activating factor (PAF) by endothelial cells. In fact, the synthesis of this potent activator of platelets and neutrophils was induced by stimulation of the same receptor subtype that induced PGI2 synthesis: stimulation of a histamine H1 or a bradykinin B2 receptor induced both PAF and PGI2 synthesis. However, two physiologically important differences exist between the production of PAF and PGI2 by endothelial cells. The synthesis of PGI2 proceeded for only 7.5 min before the abrupt termination of synthesis, whereas the synthesis of PAF was clearly detectable even 45 min after stimulation. Although maximal accumulation of PAF occurred after 10-15 min of stimulation, the prolonged synthesis resulted in the presence of PAF for up to 1 h after stimulation. Secondly, whereas PGI2 was released from the cell monolayer, PAF remained cell-associated without significant release to the external medium. Endothelial cell-generated PAF, therefore, does not function as a hormone. The prolonged association of this potent activator of platelets and neutrophils with endothelial cells may mediate some of the inflammatory properties of histamine and bradykinin. It may also be a factor in the formation of a thrombogenic vascular surface, an event suggested to play a primary role in the pathogenesis of thrombosis and atherosclerosis.     

Sch 37370: a potent, orally active, dual antagonist of platelet-activating factor and histamine

Platelet-activating factor (PAF) and histamine are potent bronchospastic agents and possess additional properties such as induction of vasopermeability and activation of inflammatory cells that are consistent with their ability to mediate allergic and inflammatory responses. From a structural series with anticipated antihistamine activity, Sch 37370 (1-acetyl-4(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2- b]pyridine-11-ylidine)piperidine) has been identified as a dual antagonist of PAF and histamine in vitro and in vivo and has been compared with several selective antagonists of PAF and histamine. Sch 37370 selectively inhibits PAF-induced aggregation of human platelets (IC50 = 0.6 microM) and also competes with PAF binding to specific sites in membrane preparations from human lungs (IC50 = 1.2 microM). Sch 37370 blocks the binding of [3H]pyrilamine to histamine-H1 receptors in rat brain membranes. Administered i.v. to guinea pigs, Sch 37370 is an equipotent antagonist of PAF and histamine-induced bronchospasm (ED50 = 0.6-0.7 mg/kg). Orally in guinea pigs, Sch 37370 is somewhat more effective against bronchospasms to histamine (ED50 = 2.4 mg/kg) than against PAF (ED50 = 4.1-6.0 mg/kg) or serotonin (ED50 = 9.6 mg/kg). Sch 37370 only weakly antagonizes methacholine-induced bronchospasm (ED50 = 51 mg/kg) and is completely inactive at 50 mg/kg against leukotriene C4 or substance P. Sch 37370 blocks hypotension in rats and a cutaneous reaction in monkeys induced by either PAF or histamine, as well as PAF-induced lethality in mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell-Mediated Immune Response of Ragweed-Sensitive Patients to Ragweed Antigen E: IN VITRO LYMPHOCYTE TRANSFORMATION AND ELABORATION OF LYMPHOCYTE MEDIATORS

The in vivo and in vitro responses to ragweed antigen E were evaluated in 28 untreated atopic patients with ragweed hayfever. The methods employed included direct skin testing, measurement of total serum IgE, measurement of specific IgE anti-ragweed antibodies, leukocyte histamine release, lymphocyte transformation, and release of lymphocyte mediators (migration inhibitory factor and mitogenic factor). The patients could be divided into sensitive and insensitive groups on the basis of their in vitro reactivity to antigen E. 20 patients in the sensitive group had statistically higher levels of total serum IgE, higher levels of specific IgE anti-ragweed antibodies, and greater leukocyte sensitivity as measured by antigen-induced histamine release than did eight patients in the insensitive group. Lymphocytes from sensitive patients produced greater amounts of migration inhibitory factor and mitogenic factor when challenged by antigen E than did lymphocytes from insensitive patients. A possible role for the lymphocyte in this allergic disease is discussed. The results of this study indicate that the immune response to ragweed antigen is complex and involves components of both immediate and delayed hypersensitivity.     

Increased release of histamine by anti-IgE from leucocytes of asthmatic patients and possible heterogeneity of IgE

A study was made of the significantly increased amounts of histamine released by anti-IgE from the leucocytes of asthmatic and allergic patients, particularly in patients with a low level of serum IgE. By measuring cell-bound IgE and histamine release with anti-IgE, particularly after passive sensitization of human leucocytes, it was possible to show that serum IgE in some allergic patients differed from that in normal subjects in two respects. Firstly, it appeared to have a relatively high leucocyte binding affinity, though this could not be proved conclusively. Secondly, and more importantly, IgE of some patients could more readily mediate histamine release with anti-IgE, that is to say it had a greater efficacy. These properties should be differentiated from another feature of allergic individuals, that is the more ready releasability of histamine from the patients' own leucocytes.     

ISOLATION AND CHARACTERIZATION OF PERMEABILITY FACTORS FROM RABBIT NEUTROPHILS

Four basic proteins that increase vascular permeability have been isolated in purified form from rabbit neutrophilic granules. These proteins are termed band 1, 2, 3, and 4 protein according to their electrophoretic migration in acrylamide gel. Molecular weights of band 1 and 2 protein derived from amino acid composition were 4800 and 5300, respectively. These values are in good agreement with those obtained for these proteins by gel diffusion techniques. The molecular weight of band 3 protein was also in the range of 5000 by the latter technique. The molecular weight of band 4 protein determined by ultracentrifugal analysis and amino acid composition was 12,000. Although all four proteins had the capacity to induce immediate increase in vascular permeability, only band 2 protein was found to release histamine from isolated rat peritoneal mast cells. Furthermore, it has been shown that the permeability-inducing activity of band 2 protein can be inhibited by pretreating rabbits with antihistamine. Band 2 protein did not release histamine from rabbit platelets and depletion of rabbit platelets from the circulation had no influence on the permeability-inducing activity of this protein. Band 1, 3, and 4 proteins did not release histamine from isolated rat peritoneal mast cells and their capacity to increase vascular permeability remained unaffected by treatment of rabbits with antihistamine. These investigations suggest that the histamine-releasing activity of band 2 protein is a specific phenomenon and is associated with particular amino acid grouping or spacial configuration of the molecules. By the same token, the increase in vascular permeability induced by the nonhistamine-releasing band 1, 3, and 4 proteins represents a specific phenomenon (or phenomena) not particularly related to the over-all charge of these molecules.     

蛤蚧定喘胶囊的平喘作用及其作用机制的实验研究

目的　探讨蛤蚧定喘胶囊 (GDC)对哮喘豚鼠的平喘作用及作用机制。方法　①采用等容积氯化乙酰胆碱和磷酸组胺雾化引喘 ,观察豚鼠给药前、后引喘潜伏期的变化。②采用 4%卵清蛋白生理盐水和4%氢氧化铝凝胶致敏 ,卵清蛋白复制哮喘模型 ,放射免疫法测定血清总IgE的含量 ;生物定量法测定血浆中血小板活化因子 (PAF)的含量。结果　①GDC给药后豚鼠的引喘潜伏期比给药前及正常组均显著延长 (P <0 0 1)。②GDC治疗组与模型组比较 ,血清总IgE含量显著降低 (P <0 0 1) ,与正常组差异无显著意义 (P >0 0 5 ) ;血浆PAF含量显著降低 (P <0 0 1) ,但仍高于正常组。结论　GDC有明显的平喘作用 ,其作用机制与其降低哮喘模型豚鼠异常升高的血清IgE水平和血浆PAF水平 ,进而参与机体的免疫调节作用有关。     

Action of platelet-activating factor on type 1 diabetic human platelets

Platelets from patients with type 1 diabetes have exhibited more sensitivity to aggregation when compared with platelets from controls without diabetes after challenge with platelet-activating factor (PAF). The production of thromboxane B2 (TxB2) and 12-hydroxyeicosatetraenoic acid (12-HETE) and the release of 5-hydroxytryptamine (5HT) were increased when the platelets were challenged by PAF (5.0 X 10(-6) mol/L and 1.0 X 10(-6) mol/L). The production of TxB2 and 12-HETE and the release of 5HT were related to the irreversible biphasic aggregation profiles observed in the patients with diabetes. Inhibition of thromboxane A2 (TxA2) production by acetylsalicylic acid abolished the secondary wave of aggregation of platelets from patients with diabetes, changing an irreversible aggregation to a reversible one. Inhibition of both TxA2 and 12-HETE production by eicosatetraynoic acid did not contribute further to the inhibition caused by acetylsalicylic acid alone, indicating that 12-HETE was not involved in the secondary wave of aggregation. These data show that the increased aggregation observed in the platelets from the group with diabetes in response to PAF results in part from their higher production of TxA2 and release of 5HT.     

Activation and desensitization of platelets by platelet-activating factor (PAF) derived from IgE-sensitized basophils. I. Characteristics of the secretory response

The secretion of vasoactive amines from rabbit platelets induced by the platelet-activating factor (PAF) derived from IgE-sensitized rabbit basophils, was examined. The secretion required calcium has previously been shown to be noncytotoxic and was optimal in both rate and extent at 37 degrees C and pH 7.2. Different temperature-sensitive steps were rate limiting for secretion above or below 20 degrees C. The rate of secretion was dependent upon the concentration of PAF and also of platelets. Maximal rates were observed with relatively low concentrations of platelets (2.5 X 10(8)/ml), sharply contrasting with other platelet stimuli such as C3 or thrombin. The extent of secretion was dependent upon PAF concentration until a maximum of 50 or 60% of the serotonin was released and then declined with increasing amounts of PAF. This was interpreted to result from the platelets becoming desensitized to the PAF, a process that shuts off the secretion. Such a desensitization was demonstrated and was shown to be stimulus specific, i.e., other stimuli could still induce secretion from PAF-desensitized platelets. PAF extracted with ethanol from the albumin to which it is usually bound during preparation, exhibited similar characteristics, except that secretion of up to 90% of the serotonin was induced. The extracted PAF thus seemed less able to induce the desensitization. Its use did provide important evidence that populations of rabbit platelets are relatively homogenous in their ability to respond to PAF.     

Histamine secretion from human skin slices induced by anti-IgE and artificial secretagogues and the effects of sodium cromoglycate and salbutamol

Human cutaneous mast cells show functional differences from their counterparts in other tissues. Following passive sensitization with 1% atopic serum for 30 min at 37 degrees C human skin slices released histamine after challenge with anti-human IgE in a concentration dependent manner. Maximum release of 14 +/- 2% was achieved with a 1/10 dilution of anti-IgE. Passive sensitization with 10% atopic serum increased the secretory response to anti-IgE but histamine release was only concentration related over the entire 1/1000 to 1/10 dilution range in half of the specimens studied, the remainder showing high dose tolerance to anti-IgE. Negligible histamine release occurred with anti-IgE challenge of slices which had not been passively sensitized. The histamine releasing ability of A23187 in human skin slices was similar to that observed in lung and adenoidal mast cells being concentration dependent over the range 0.1-3 microM with a maximum release of 25 +/- 3%. In contrast to human lung and adenoidal mast cells, poly-L-lysine and compound 48/80 induced histamine release from skin slices. Poly-L-lysine induced a concentration-dependent release of histamine over the range 0.01-10 microM with a maximum of 27 +/- 3%. The response to compound 48/80 was variable, releasing in some but not all specimens. Histamine release caused by anti-IgE, A23187 and poly-L-lysine was shown to be dependent upon extracellular calcium while release stimulated by compound 48/80 was calcium independent. The chemotactic peptide, formyl-methionyl-leucyl-phenylalanine, over the range 0.01-10 microM failed to release histamine from skin slices.(ABSTRACT TRUNCATED AT 250 WORDS)     

IgE Antibody Measurements in Ragweed Hay Fever RELATIONSHIP TO CLINICAL SEVERITY AND THE RESULTS OF IMMUNOTHERAPY

Specific IgE anti-ragweed antibodies (IgEAR) were measured over two years in two groups of highly sensitive patients treated (immunized) with either ragweed extract or placebo and in a third group of placebo-treated, relatively insensitive patients. The IgEAR on the patients' basophils were assessed by ragweed antigen E (AgE)-induced histamine release; blocking (IgG) antibodies were measured by their ability to inhibit AgE induced histamine release. These data were evaluated against the clinical severity of ragweed hay fever in each patient.WE FOUND THAT: (a) In placebo treated patients IgEAR usually declined gradually prior to the ragweed season and were boosted by environmental exposure to ragweed pollen. (b) In immunized patients the IgEAR rose at the beginning of treatment, but fell as immunotherapy proceeded; by the end of the second year the levels had decreased in 18/19 patients. (c) The increase in blocking antibody during immunotherapy correlated significantly (P < 0.05) with the decrease in serum IgEAR. (d) Judged by their sensitivity to AgE induced histamine release, IgEAR on basophils correlated significantly with IgEAR in the serum of untreated patients (P < 0.01). (e) The highly sensitive placebo treated patients' symptom scores were significantly correlated with their IgEAR in serum (P < 0.01) and with the sensitivity of their basophils to AgE-induced histamine release (P < 0.01). Neither correlation was observed in the relatively insensitive patients. (f) In the treated group the IgEAR measurements predicted neither the degree of their illness nor their clinical improvement We conclude tha IgE antibody measurements may be useful in the assessment of the severity of reaginic allergy in highly sensitive patients. Its use in modestly sensitive patients requires patients requires further study, as does the inverse association between IgE and IgG antiragweed antibodies.     

Response to platelet-activating factor in human platelets stored and aged in plasma. Decrease in aggregation, phosphoinositide turnover, and receptor affinity

Human platelet concentrates were stored in polyolefin bags at 22 to 24 degrees C on a horizontal shaker for up to 8 days. At different intervals, aliquots of platelet-rich plasma (PRP) were removed aseptically and five variables, i.e., platelet counts, morphology, platelet-activating factor (PAF)-stimulated aggregation, phosphoinositide turnover, and [3H]PAF binding to platelet receptors, were studied. The number of platelets did not change during the 8 days of storage. Scanning electron microscopy of the platelets revealed a gradual morphologic change from biconcave flat discs to irregular, crenated forms. The PAF-induced aggregation of platelets declined with time of storage. A decrease to 50 percent of the Day 1 aggregatory response to PAF was evident on Day 2, and there was a further decline to about 20 percent by Day 6. Similarly, PAF receptor-coupled phosphoinositide turnover, as monitored by 32P incorporation into individual phosphoinositides, decreased dramatically with storage. After 2 to 3 days of storage, the phosphoinositide turnover was reduced to 50 percent of the original response, and it continued to decline to about 25 percent of original response by Day 5 or 6. The binding of [3H]PAF to washed human platelets indicated subtle changes between Days 2 and 4, which became more noticeable by Day 6. These results have raised the possibility of changes in the number of the receptors and/or their affinity for the ligand during storage. We conclude that although the number of platelets was maintained during storage for 8 days, a general deterioration of their responses to PAF occurred at the levels of cell surface receptor, transmembrane signaling (phosphoinositide turnover), and response (aggregation).     

CV-6209, a highly potent antagonist of platelet activating factor in vitro and in vivo

2-[N-acetyl-N-(2-methoxy-3-octadecylcarbamoyloxypropoxycarbonyl) aminomethyl]-1-ethylpyridinium chloride (CV-6209) inhibited aggregation of rabbit and human platelets induced by platelet activating factor (PAF) with the IC50 values of 7.5 X 10(-8) and 1.7 X 10(-7) M, respectively, and had little effects on the aggregation induced by arachidonic acid, ADP and collagen. The inhibitory effect of CV-6209 on the PAF-induced rabbit platelet aggregation was 104, 9, 8 and 3 times more potent than the PAF antagonists CV-3988, ONO-6240, Ginkgolide B and etizolam, respectively. CV-6209 inhibited [3H]serotonin release from rabbit platelets stimulated with PAF (3 X 10(-8) M) with a similar potency as the inhibition on the platelet aggregation. CV-6209 inhibited PAF (0.3 microgram/kg i.v.)-induced hypotension in rats (ED50, 0.009 mg/kg i.v.) with no effect on the hypotension induced by arachidonic acid, histamine, bradykinin and isoproterenol. CV-6209 (1 mg/kg) inhibited slightly the acetylcholine-induced hypotension. In rats, post-treatment with CV-6209 reversed the PAF (1 microgram/kg i.v.)-induced hypotension rapidly (ED50, 0.0046 mg/kg i.v.); CV-6209 was 74, 20, 185 and over 2100 times more potent than CV-3988, ONO-6240, Ginkgolide B and etizolam, respectively. Thus, the relative potency of the anti-PAF action of PAF analog (CV-6209, CV-3988 and ONO-6240) differed little between the inhibition of PAF-induced platelet aggregation and the reversal of PAF-induced hypotension, but that of nonPAF analogs (Ginkgolide B and etizolam) differed greatly with these assay systems, when standardized with CV-6209.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunologic and cellular changes accompanying the therapy of pollen allergy

We had found previously that children with ragweed hay fever were somewhat less symptomatic after preseasonal immunization with large doses of ragweed pollen extract than were placebo-treated children. To study further the immunologic changes which accompany immunotherapy, these children were treated again the following year. Each patient served as his own control.Serum blocking (IgG) antibody, measured by inhibition of antigen-induced leukocyte histamine release, was increased 20- to 40-fold after therapy. The anticipated postpollen season increase of serum reaginic (IgE) antibody, measured by passive sensitization of leukocytes from nonallergic donors, was suppressed. Instead, the mean titer was decreased after treatment. Total serum IgE levels, measured by radial radioimmunodiffusion assay, were higher than normal; were correlated with reaginic antibody titers; and also did not increase in the pollen season after treatment. The concentration of both IgE and reaginic antibody was lower in the older children, irrespective of treatment.Leukocyte response to ragweed antigen E and guinea pig anti-IgE antiserum was assessed by means of in vitro histamine release techniques. After treatment, the leukocytes of 21 patients were less sensitive (11 cases), or less reactive (10 cases), to antigen E. Response to anti-IgE antibody also was diminished after treatment. In four cases, neither anti-IgE nor antigen E induced histamine release, although both IgE protein and ragweed-specific IgE antibody were present in the patients' own sera.Clinical improvement was correlated best with decreased leukocyte sensitivity and leukocyte reactivity to ragweed antigen E. It appeared that decreased cell sensitivity was related to lower serum reaginic antibody levels. Decreased cell reactivity, in the presence of both IgE protein and IgE antibody in the serum, may indicate a change in cellular response mechanisms. These studies suggest that clinical improvement following specific immunotherapy must be the result of complex changes in the immunologic and cellular components of allergic disease.     

Characterization of serum platelet-activating factor (PAF) acetylhydrolase. Correlation between deficiency of serum PAF acetylhydrolase and respiratory symptoms in asthmatic children

Platelet-activating factor (PAF) acetylhydrolase has been recognized as an enzyme that inactivates PAF. We developed a convenient and reproducible method for determining human serum PAF acetylhydrolase activity. The assay was based on measurement of [14C]acetate produced from 1-O-alkyl-2-[14C]-acetyl-sn-glycero-3-phosphocholine upon precipitation of the complex of radioactive substrate and albumin with TCA. The apparent Km value of PAF acetylhydrolase (near the physiological concentration of serum protein) was 1.5 X 10(-4) M PAF. 32 subjects with serum PAF acetylhydrolase deficiency were found among 816 healthy Japanese adults. The low PAF acetylhydrolase activity in the deficient serum might not be due to the presence of enzyme inhibitor. Both the sensitivity to PAF and the metabolism of PAF in platelets from PAF acetylhydrolase-deficient subjects were almost the same as those of normal subjects. Deficiency in serum PAF acetylhydrolase appeared to be transmitted by autosomal recessive heredity among five Japanese families. Among healthy adults, healthy children, and asthmatic children, who were grouped into five classes on the basis of respiratory symptoms (remission, wheezy, mild, moderate, and severe groups), the probability of PAF acetylhydrolase deficiency was significantly higher in groups with severe symptoms (moderate and severe) (P less than 0.01). These results suggest that deficiency of serum PAF acetylhydrolase might be one of the factors leading to severe respiratory symptoms in asthmatic children.     

A lavage method for dynamic intraarticular monitoring of animal joints in situ: quantification and release kinetics of histamine after selective synovial mast cell activation by diverse secretagogues.

Evidence implicating synovial mast cells in the initiation and perpetuation of arthritis has increased. We have developed a method of joint lavage to monitor dynamic intraarticular events in the intact animal. Lavage was done before and after immunologic (passive sensitization followed by intravenous specific antigen or intraarticular anti-rat immunoglobulin E [IgE] heteroantiserum) and nonimmunologic (intraarticular calcium ionophore A-23187; phorbol 12-myristate, 13-acetate; and compound 48/80) synovial mast cell activation. To quantify and analyze synovial mast cell mediator release kinetics in situ, we measured lavage fluid histamine. With all activation protocols except A-23187, histamine release was evident within 5 minutes after introduction of the stimulus. The quantitative and chronological similarities between immunologically induced and compound 48/80-induced synovial mast cell histamine release kinetics suggested that connective tissue type mast cells are an important source of inflammatory mediators in rat joints. We also measured joint lavage fluid histamine levels in rats immunized with an active sensitization protocol. Histamine levels were determined by the autoanalyzer method and were confirmed by using a commercially available radioimmunoassay that uses a monoclonal antibody against acetylated histamine. We found that in many of these animals, at the peak of the serum IgE response, joint lavage fluid histamine levels were very high even before challenge with specific antigen, and that this increase was not due to diffusion into the joint of abnormally elevated plasma histamine. These data suggested that synovial mast cells are preferentially activated in states of high serum IgE immune responses. We have used a simple, inexpensive, rapid lavage technique to generate the first data on histamine release kinetics after selective synovial mast cell activation in the intact animal. The technique can be adapted for investigation of release kinetics of a variety of other substances from activated synovial cells and can be used in other arthritis models.     

Interaction of Ca2+ and protein phosphorylation in the rabbit platelet release reaction.

Ca2+ flux and protein phosphorylation have been implicated as playing an important role in the induction of the platelet release reaction. However, the interactions between Ca2+, protein phosphorylation, and the release reaction have been difficult to study because secretion in human platelets is independent of extracellular Ca2+. Thus, we studied rabbit platelets, which, unlike human platelets, require extracellular Ca2+ for serotonin release to occur. Thrombin, basophil platelet-activating factor (PAF), or ionophore A23187 treatment of intact 32PO43--loaded rabbit platelets resulted in a 200-400% increase in phosphorylation of P7P and P9P, respectively. These peptides were similar in all respects to the peptides phosphorylated in thrombin-treated human platelets. When Ca2+ was replaced in the medium by EGTA, (a) thrombin- and PAF-induced rabbit platelet [3H]serotonin release was inhibited by 60-75%, whereas ionophore-induced release was blocked completely; (b) thrombin-, PAF-, or ionophore-induced P9P phosphorylation was inhibited by 60%; and (c) ionophore-induced P7P phosphorylation was decreased by 60%, whereas that caused by thrombin or PAF was decreased by only 20%. At 0.25-0.5 U/ml of thrombin, phosphorylation preceded [3H]serotonin release with the time for half-maximal release being 26.0 +/- 1.3 s SE (n = 3) and the time for half-maximal phosphorylation being 12.3 +/- 1.3 s SE (n = 3) for P7P and 3.7 +/- 0.17 s SE (n = 3) for P9P. P9P phosphorylation was significantly inhibited (P less than 0.015) by removal by Ca2+ from the medium at a time point before any thrombin- or ionophore-induced serotonin release was detectable. Thus, our data suggest that Ca2+ flux precedes the onset of serotonin secretion and that the rabbit platelet is an appropriate model in which to study the effects of Ca2+ on protein phosphorylation during the platelet release reaction.     

Histamine release from human leukocytes: studies with deuterium oxide, colchicine, and cytochalasin B

Agents known to interact with either microtubules or microfilaments influenced the antigen-induced release of histamine from the leukocytes of allergic individuals. Deuterium oxide (D₂O) which stabilizes microtubules and thereby favors their formation enhanced histamine release markedly. Concentrations as low as 5% increased antigen-induced release somewhat while concentrations as high as 75% had no effect on release in the absence of antigen. Enhancement occurred over a wide range of antigen concentrations and was also seen when release was initiated by antibody to IgE or IgG. When the release process was divided into two stages a D₂O activity could be demonstrated only in the second stage. However, when D₂O was present in the first stage together with agents which raise cyclic AMP levels and thereby inhibit release it partially reversed this inhibition. Colchicine, demecolcine, and vinblastine, compounds known to disaggregate microtubules, i.e., have an effect opposite to that of D₂O, inhibited the release of histamine and counteracted the effects of D₂O. The inhibitory action of colchicine was greater if cells were treated with colchicine before rather than after activation with antigen. Cytochalasin B, a compound which causes the disappearance of microfilaments, had variable effects on histamine release. The most frequently seen response was slight enhancement. Neither D₂O nor cytochalasin B altered cyclic AMP levels in leukocytes. These observations support and strengthen the view that an intact and functioning microtubule system is directly important for the secretion of histamine from leukocytes and suggest that microfilaments might have multiple indirect effects.     

Histamine regulates cytokine production in maturing dendritic cells, resulting in altered T cell polarization

Atopic diseases such as allergy and asthma are characterized by increases in Th2 cells and serum IgE antibodies. The binding of allergens to IgE on mast cells triggers the release of several mediators, of which histamine is the most prevalent. Here we show that histamine, together with a maturation signal, acts directly upon immature dendritic cells (iDCs), profoundly altering their T cell polarizing capacity. We demonstrate that iDCs express two active histamine receptors, H1 and H2. Histamine did not significantly affect the LPS-driven maturation of iDCs with regard to phenotypic changes or capacity to prime naive T cells, but it dramatically altered the repertoire of cytokines and chemokines secreted by mature DCs. In particular, histamine, acting upon the H2 receptor for a short period of time, increased IL-10 production and reduced IL-12 secretion. As a result, histamine-matured DCs polarized naive CD4(+) T cells toward a Th2 phenotype, as compared with DCs that had matured in the absence of histamine. We propose that the Th2 cells favor IgE production, leading to increased histamine secretion by mast cells, thus creating a positive feedback loop that could contribute to the severity of atopic diseases.     

ACUTE IMMUNE COMPLEX DISEASE IN RABBITS : THE ROLE OF COMPLEMENT AND OF A LEUKOCYTE-DEPENDENT RELEASE OF VASOACTIVE AMINES FROM PLATELETS

By depletion of C3 from rabbits undergoing acute experimental immune complex disease with an anticomplementary factor in cobra venom, it has been possible to demonstrate that deposition of the complexes in arteries and glomeruli does not require the complement components reacting after C2. Immunological reactions, in which platelets release their vasoactive amines, have been examined in rabbits undergoing immune complex disease. A correlation was obtained between the presence of a complement-independent reaction which required blood leukocytes, antigen and platelets, the deposition of immune complexes, and the induction of glomerulonephritis. C3 depletion did, however, have a marked alleviating effect on the severity of the arterial lesions. Neutrophil accumulation and the subsequent necrotizing arteritis were prevented. In contrast, the character and severity of the glomerulonephritis was not altered by depletion of later-acting complement components.