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With Deuterium Oxide, Colchicine, and Cytochalasin B

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With Deuterium Oxide, Colchicine, and Cytochalasin B Histamine Release from Human Leukocytes: Studies with Deuterium Oxide, Colchicine, and Cytochalasin B ELIZABETH GLLESPIE and LAWRENCE M. LICHTENSTEIN From the Division of Clinical Immunology, The Johns Hopkins University School of Medicine at The Good Samaritan Hospital, Baltimore, Maryland 21239 A B S T R A C T Agents known to interact with either INTRODUCTION microtubules or microfilaments influenced the antigen-in- Antigen-mediated release of histamine from the duced release of histamine from the leukocytes of al- leuko- lergic individuals. Deuterium oxide (D20) cytes of allergic individuals is a good in vitro model for which sta- allergic reactions. The extent of bilizes microtubules and thereby favors their formation release correlates well enhanced histamine release with clinical symptoms (1) and drugs, e.g., isoproterenol markedly. Concentrations and theophylline, which alleviate the as low as 5% increased antigen-induced release somne- in vivo symptoms also inhibit the in vitro release what while concentrations as high as 75% had no effect process (2). At the same time the mechanism by which histamine is released from on release in the absence of antigen. Enhancement oc- leukocytes curred over a wide range of antigen is fundamentally similar to the release process concentrations and in other tissues, with cell-bound was also seen when release was initiated by antibody (IgE) acting antibody to as a IgE or IgG. When the release process was divided into receptor and antigen replacing a hormone or other two stages a D20 activity could be demonstrated only releasing agent. in the second stage. However, when D20 was present in Colchicine, a drug known to disrupt microtubules, has the first stage together with agents which raise cyclic been shown to inhibit the release of a variety of com- AMP levels and thereby inhibit release it partially re- pounds (3-6) including histamine from leukocytes (7). versed this inhibition. Colchicine, demecolcine, and vin- This observation has been interpreted in all cases as blastine, compounds known to disaggregate microtubules, indicating that microtubules play a role in the release i.e., have an effect opposite to that of D20, inhibited the process. Deuterium oxide (D20) which is known to sta- release of histamine and counteracted the effects of D20. bilize microtubules (8, 9), i.e., act in a manner opposite The inhibitory action of colchicine was greater if cells to colchicine, has been studied in three release systems. were treated with colchicine before rather than after In the rat peritoneal mast cell it potentiates the release activation with antigen. Cytochalasin B, a compound of histamine by other releasing agents and also acts as a which causes the disappearance of microfilaments, had releasing agent itself (3); in the cat adrenal gland it variable effects on histamine release. The most fre- potentiates the release of catecholamines by acetylcholine quently seen response was slight enhancement. Neither (6); and in the thyroid gland it inhibits release (10). D20 nor cytochalasin B altered cyclic AMP levels in leu- Recently cytochalasin B, a drug which causes the disap- kocytes. These observations support and strengthen the pearance of 40-70 A microfilaments from cells but has no view that an intact and functioning microtubule system effect on the morphology of microtubules (11, 12) has is directly important for the secretion of histamine from been shown to inhibit the release of thyroid hormone leukocytes and suggest that microfilaments might have and growth hormone (13, 14) and potentiate the release multiple indirect effects. of insulin (15). The present study was designed to evaluate the role This work was presented in part at the 56th Annual Meet- of microtubules and microfilaments in the release of his- ing of the Federation of American Societies for Experi- tamine from leukocytes through the use of D20, colchi- mental Biology and appeared in abstract form in 1972 Fed. Proc. 31: 748. cine, and cytochalasin B individually and in combination. Received for publication 2 June 1972 and in revised form It was found that D20 potentiated the release process 18 July 1972. markedly while colchicine and other microtubule dis- The Journal of Clinical Investigation Volume 51 November 1972 2941 rupters inhibited release and counteracted the effect of 100 - D20. Cytochalasin B had variable effects depending on the individual cells used and the level of histamine re- 80 - lease obtained. In the majority of cases it potentiated 60. release somewhat although inhibition was occasionally 0 50% U) observed. These results support the view that micro- '< 40 l tubules are directly important in the release process and w suggest that microfilaments have multiple indirect effects. I;20 z D20 0 METHODS Leukocytes from allergic individuals were prepared by dex- I AN>TI -IgE (qg/ml ) ,- 100 tran sedimentation as previously described (16). They were z resuspended at a dilution of approximately 107 cells/ml and incubated at 37'C in a medium consisting of (mM): NaCl, a: D2 =56 120; KCl, 5; CaCl2, 0.6; MgC12, 1; tris (hydroxymethyl) aminomethane, 25; human serum albumin, 0.1%, adjusted 60 to pH 7.4. D20 replaced H20 in the medium as required. Experiments involving colchicine were also carried out 40 Xf~~~~~ using a phosphate buffer since tris has been reported to interfere with the action of colchicine (17). Under our 20 - conditions the effect of colchicine in the two buffers was I - -11 the same. In experiments designed to study the two stages o I I of the release process (18) cells were exposed to antigen - -6 AIG4 1Em for 2 min in medium free of calcium and magnesium, ANTIGENs E ( ug/ml ) washed twice and resuspended in the absence of antigen in the complete medium described above. Histamine released FIGURE 2 The effect of D20 on histamine release induced from the cells into the supernatant fluid was measured by by either antigen E or anti-IgE antibody. Releasing agent a micromodification of the fluorometric technique of Shore and D20 were added at 0 time and the cells incubated for (19, 20). All experiments were carried out in duplicate 45 min. and repeated using the cells of at least two and usually three to five different individuals. The average difference protein prepared from bovine adrenal cortex. Charcoal is between duplicate measurements using this technique was used to separate free and bound nucleotide. less than 5%. Ragweed antigen E was provided by Dr. T. P. King Cyclic AMP was measured by the protein-binding method (22) and rye grass group I antigen by Dr. D. G. Marsh of Brown, Albano, Ekins, and Sgherzi (21). This proce- (23). D20 was obtained from BioRad Laboratories (Rich- dure is based on the competition between cyclic AMP in mond, Calif.), demecolcine (Colcemid) from Ciba Pharma- the sample and added radioactive cyclic AMP for a binding ceutical Co. (Summit, N. J.), vinblastine sulfate (Velban) from Eli Lilly & Co. (Indianapolis, Ind.), and cytochalasin B from 100 Antigen = 1.4xlO pg/ml Imperial Chemical Industries (Alderly Park, Eng- land). This last drug was dissolved in dimethylsulfoxide w (DMSO) (10 mg/ml) and diluted as required with incuba- U)LJ tion medium. DMSO controls were included in all experi- 80 -5 J 1 Antigen=- x 10 jpg/ml ments involving cytochalasin B. z RESULTS I 60 Effects of DAO on histamine release. D20 markedly U) enhanced the release of histamine from leukocytes under m a variety of experimental conditions. D20 at levels as z w 40 low at 5% significantly increased release due to antigen while as w concentrations high as 75% had no effect in the a- absence of antigen (Fig. 1). Enhancement occurred 20 over a wide range of antigen concentrations including levels insufficient to cause release alone and levels high NoAntigen enough to inhibit the process (Fig. 2). Enhancement of anti-IgE mediated release (24) was similar in all re- 20 40 60 spects to that noted with antigen (Fig. 2). Potentiation PER CENT D20 of release by D20 was also seen using the cells of indi- FIGURE 1 The effect of various concentrations of D20 on viduals who can maximally release only 10-20% of their histamine release. Ragweed antigen E and D20 were added total histamine and in situations where the releasing at 0 time and the cells incubated for 45 min. agent usually can induce only a limited response, i.e., 2942 E. Gillespie and L. M. Lichtenstein antibodies to IgG (25) (Fig. 3). When the time course TABLE I of release was studied, D20 increased the rate of re- Effects of D 0 on Histamine Release from the Leukocytes lease rather than otherwise altering the time course (re- of Various Individuals sults not shown). Enhancement of histamine'release by Control Potentiation by D20 D20 of the order of magnitude illustrated in Figs. 1-3 histamine was seen in experiments with the cells of all individuals Subject release D20: 7% 15% 31% 62% studied (Table I). The per cent enhancement is, of course, related to control release in that if release in the % ~~% % % % M. C. 38* 85 absence of D20 is 50% enhancement cannot exceed 100% M. C. 56 42 while if control release is 5% enhancement can be P. A. 34 35 182 2000%. B. W. 14 100 321 414 The release of histamine from leukocytes can be op- B. W. 24 58 158 316 erationally divided into two stages, an antigen-dependent, J.S. 4 1100 calcium-independent first stage and an antigen-inde- J. S. 34 162 pendent, calcium-dependent second stage (18). The ef- C. S. 5 520 fects of D20 on the two stages of the release reaction M. M. 13 154 K. B. 40 85 were studied by carrying out either the first or the sec- S.
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