1236

BIOCHEMICAL SOCIETY TRANSACTIONS

4. Crompton, M. R., Moss, S. E. & Crumpton, M. J. ( 1 988) Cell (Chmhridge, Muss) 5 5 , 1-3 5. Haigler, H. T.,Fitch, J. M., Jones, J. M. & Schlaepfer, D. D. ( 1989)Trends Biochem. Sci. 14,48-50 6. Klee. C. B. ( 1988)Biochemistry 27,6645-6653 7. Davidson, F. F., Dennis, E. A,, Powell, M. & Glenney, J. R. (lY87)J.B i d . Chem. 262,1698-1705 8. Haigler, H. T., Schlaepfer, D. D. & Burgess, W. H. (1987)J. Biol. Chem. 262,6921-6930 9. Glenney, J. R.( 1987)BioEssuys 7, 173- 175 10. Sudhof, T. C., Ebbecke, M., Walker, J. H., Fritsche, U. & Boustead, C. M. (1984)Biochemistry 23,1103-1 109 1 1 . Geisow, M. J., Fritsche, U., Hexham, J. M., Dash, B. & Johnson, T. ( 1 986)Nature (London) 320,636-638

12. Creutz, C. E., Zaks, W. J., Hamman, H. C., Crane. S.. Martin, W. H., Could, K. L., Oddie, K. M. & Parsons, S. J. (1987)J. )Biol.Chem. 262, 1860-1868 13. Geisow, M.J. & Burgoyne, R. D. (1987)Ann. N. Y. Acud. Sci. 493,563-576 14. Shadle, P. J. & Weber, K. ( 1987)Biochim. Biophys. ACIU897, 502-506 15. Braslau, D. L.,Ringo, - D. L. & Rocha, V. ( 1984)Exp. Cell Hes. 155,213-221 16. McKanna, J. A. & Cohen, S. (1985)J. Cell Biol. 101,300a

Received 12 June 1990

Localization of lipocortin-1 in rat hypothalamus and pituitary gland MARY D. WOODS,* J 6 Z S E F Z . KISS,? T E Z SMITH,$ JULIA C. BUCKINGHAM,$ ROD FLOWERS and FERENC A. ANTONI* *M.R.C.Brain Metabolism Unit, Edinburgh EH8 9JZ, U.K., t Department de Morphologie, University of Geneva, Switzerland, CHI21I ; $Department of Pharmacology, C'haring Cross and Westminster Hospital Medical School, London W6 8RF, U . K . and §Department of Biochemical I'harmacology, St. Bartholomew S Medical College, London ECI, U.K. Lipocortin-1 is a 35 kDa member of the highly conserved and widely distributed family of Ca*+ and phospholipidbinding proteins collectively known as annexins o r lipocortins [ 11. It has been proposed that lipocortin-1 is induced by glucocorticoids to mediate the suppressive actions of steroids on cellular functions in macrophages and neutrophil granulocytes [2]. Because the secretagogue-stimulated release of adrenocorticotrophic hormone (ACTH) by anterior pituitary cells is also inhibited by glucocorticoids through the induction of new mRNA and protein [3]it was of interest to examine whether lipocortin-1 is expressed in anterior pituitary cells. Glucocorticoids are also known to suppress the secretion and synthesis of hypothalamic corticotrophin-releasing factors [4], therefore the hypothalamus was also examined as a possible site of lipocortin-1 production. Light-microscopic immunocytochemical studies were carried out on cryostat sections of rat brain and pituitary gland fixed with Zamboni's solution as well as on the ACTHproducing pituitary tumour cell line, AtT20, fixed with ethanol. For electronmicroscopy tissues were processed as described by Kiss [5]. The polyclonal antiserum AS842 [6] and the monoclonal antibody MAB 105 [ 7 ] , raised against human recombinant lipocortin-1 were used at a dilution of 1 :1000. In the brain, positive staining in the ependymal lining of the third ventricle including tancytes was found. No distinct neuronal labelling was observed except for a few ill-defined cells (possibly glia) in the medial parvicellular paraventricular nucleus of the hypothalamus. In the anterior pituitary gland a large number of hormone-producing cells stained positively with the polyclonal antiserum, and the immunostaining was localized to secretory granules of cells appearing to be mammotrophs or somatotrophs (Figs. l a & l b ) . Abbreviation used: ACTH, adrenocorticotrophic hormone.

x

Fig. 1. Irnrnunocytochemical demonstration of lipocortin-1 like material (a) rat adenohypophysis AS842, original magnification 200 x ; (b) rat adenohypophysis AS842, original magnification 38000 x , note immuno-gold labelled cell with large granules and lack of labelling in adjacent cell with small secretory granules and lack of labelling in adjacent cell with small secretory granules; (c)AtT20/D1 cells in culture, after cell sorting for large cells MAB 105, original magnification 200 x ; (d) rat peritoneal cells MAB 105, original magnification 200 x . 1990

635th MEETING, ABERDEEN However, the monoclonal antibody failed to show this reaction. No change in the pattern of immunoreactivity was found in adrenalectomized rats or adrenalectomized rats injected with dexamethasone ( 1.25 mg/kg, intraperitoneally) 2 h prior to sacrifice. The intermediate lobe contained fibrocyte-like cells with numerous processes which were immunopositive with both AS842 and MAB105. In the neurohypophysis, axonal varicosities as well as swellings (Herring bodies) were clearly labelled with AS842, although less intensively than cells in the anterior lobe. Once more, this staining occurred exclusively above secretory vesicles, but was not seen with MABl05. All immunostaining with AS842 or MAB105 was blocked after overnight preabsorption with human recombinant lipocottin- 1 (50 pmol/l). The AtT20/D1 cell line comprises a heterogeneous population of cells with regard to size. The largest of the cells stained very strongly (Fig. 1 c), while smaller cells stained less intensely and non-uniformly. Other strains of AtT20 cells, 16/16 and 16v also stained positively for lipocortin-1, the distribution of label was much more uniform and less intensive than in D 1 cells. Finally, cells prepared by peritoneal lavage from female Wistar rats were intensely immunopositive using M A B l 0 5 (Fig. 1d). Western blots of normal rat anterior pituitary gland with AS842 showed two immunoreactive bands. One co-migrated around 35 kDa with human recombinant lipocortin whilst the other was of lower molecular mass (approx. 22 kDa). The MAB105 detected only the 35 kDa band. Western blots of AtT20/Dl cells with AS842 or MAB105 gave one immunoreactive band co-migrating with recombinant human

1237 lipocortin- 1. Rat hypothalami contained only the 35 kDa material using AS842, and no changes in intensity were found upon adrenalectomy or adrenalectomy and treatment with dexamethasone. In summary, the present data show that a 22 kDa protein confined to secretory vesicles in rat adenohypophysis crossreacts with lipocortin-1 antiserum 842 and d o not support a role for lipocortin-1 in the glucocorticoid inhibition of pituitary ACTH secretion or hypothalamic corticotrophinreleasing factor production. In addition, these data suggest that transformed pituitary corticotrophs express lipocortin- 1 whilst normal corticotrophs d o not. We thank Dr R. Blake Pepinsky (Biogen, Cambridge, MA, U.S.A.), for generously supplying AS842, MAB 105 and recombinant human lipocortin. 1. Burgoyne, R. D. & Geisow, M. J. ( 19x9) Cell Calcium 10, 1 - 10 2. Flower, R. J. ( 1 9 8 8 ) Hr. J . I'hurmucol. 94,987- 10 15 3. Buckingham, J. C. & Hodges, J. R. ( I 977) J . Endocrinol. 72, 187-193 4. Dayanithi, G. & Antoni, F. A. ( 1989) Endocrinology (Baltimore) 125,308-3 I3 5. Kiss, J. Z. ( I 989) J . Neltrosci. Methods 30,29-32 6 . Pepinsky, R. B., Tizard, R., Mattaliano, R. J., Sinclair, L. K., Miller, G. T., Browning, J. L., Chow. E. P., Burne. C., Pratt, I)., Wachter, L., Hession, C., Frey, A. Z. & Wallner, B. P. (1988)J. Biol. Chem. 263. 10799- 1 0 8 I 1 7. Pepinsky, R. B., Sinclair, L. K., Dougas, I., Liang, C.-M., Lawton, P. & Browning, J. L. ( 1990) FEBS lxtt. 261, 247-252 ~~

Received 10 July I990

Characteristics of lipocortin 1 binding to the surface of human peripheral blood leucocytes NICOLAS J. GOULDING,* PAN LUYINGI. and PAUL M. GUYREt *Bath ltistitirtefor Rheirmatic Diseases, Trim Bridge, Bath RAI I M I ) , U . K . and tnepartmetit of Physiology, Dartmoiith Medical School, Iianover N i t 03756 U.S.A. Study o f the distribution and functions of the lipocortin/ calpactin family of proteins spans diverse and extensive areas of cell biology [I]. Anti-inflammatory effects of recombinant human lipocortin 1 have been reported in rat paw oedema models (21 and there is evidence for production of the molecule by human monocytes in response to corticosteroid 13, 4J. If lipocortin 1 production and release is significant in mediating the anti-inflammatory effects of corticosteroids, it is of seminal importance to characterize extracellular interactions of the molecule with cells involved in the inflammatory response. We have previously described an effect it1 v i m of recombinant human lipocortin 1 in inhibiting IgG binding to Fc, receptors on human leucocytes [5]. In this series o f experiments we report the existence of specific, saturable binding sites for exogenous lipocortin on the surface of human peripheral blood monocytes, the presence of which appear closely related to its ability to inhibit IgG binding to the type I Fc, receptor. Recombinant lipocortin 1 binding to human leucocytes was investigated using a specific monoclonal antibody (mAb) 1-B 161. Peripheral blood leucocytes were isolated by singlestep density gradient centrifugation over ficoll-hypaque M85 Abbreviations used: mAb. monoclonal antibody; FITC, fluorescein isothiocyanate.

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[7]. Mononuclear and polymorphonuclear leucocyte populations were recombined in equal proportions at a final concentration of 2 x 10" cells/ml in RPMl 1640 and subsequently incubated with 0-10 pg/ml lipocortin 1 for 1 h at 4°C or 0-30 pg/ml lipocortin 1 for 1 h at 37°C. After reaction with a supramaximal concentration of mAb I-B at 4"C, the cells were stained with FITC-conjugated F(ab'), anti-mouse IgG. Following fixation, cell surface fluorescent staining was analysed using an Ortho 50H cytofluorograph. FITC-equivalent binding sites per cell were estimated using standard fluorescein beads for calibration (Flow Cytometry Standards Corp. North Carolina, U.S.A.). Specific, saturable lipocortin 1 binding sites with 4°C maximal binding at 2 pg/ml lipocortin 1 and 37°C maximal binding at 15 pg/ml lipocortin 1 were demonstrable. Fig. 1 shows typical 37°C and 4°C binding curves from two individuals, highlighting the difference in numbers of binding sites between leucocyte populations. With 37°C lipocortin 1 preincubation, monocytes from three individuals expressed a range of 1 1 000-60 000 binding sites per cell; consistently higher than neutrophils (3500 to 2 1 000). Lymphocytes showed minimal binding. Studies on four subjects with 4°C pre-incubation revealed maximal binding of 22 500 to 142500 sites for monocytes; 15000 to 110000 sites for neutrophils and 600 to 5000 sites for lymphocytes. Interaction of lipocortin 1 with all cell types could be abrogated by the presence of 1 mM-EDTA o r the absence of calcium in the wash buffer. Surface binding oflipocortin 1 to UY37 cells and monocytes cultured for 24 h or longer was minimal. The specific interaction of lipocortin 1 with the monocyte cell surface membrane correlated directly with its ability to

Localization of lipocortin-1 in rat hypothalamus and pituitary gland.

1236 BIOCHEMICAL SOCIETY TRANSACTIONS 4. Crompton, M. R., Moss, S. E. & Crumpton, M. J. ( 1 988) Cell (Chmhridge, Muss) 5 5 , 1-3 5. Haigler, H. T.,...
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