Migration of lymphocytes across specialized vascular endothelium. V. Production of a sulphated macromolecule by high endothelial cells in lymph nodes

Sephadex G- 0 1 0 University of Manchester Medical School , Oxford Road, Manchester Mi 3 gPT, U.K 1 Experimental Pathology Laboratory and Department of Medical Biochemistry P. ANDREWS, D. W. MILSOM AND W. L. FORDf SUMMARY High endothelial cells lining the post capillary venules in the paracortical areas of rat lymph nodes were found by autoradiography to incorporate [asS]sulphate, whether it was injected into the footpad to reach the draining popliteal lymph node or added to short-term cultures of cervical lymph node slices. The early localization of [s'S]sulphate was confined to the Golgi apparatus, but before it disappeared from the cell radioactivity was associated with cytoplasmic vesicles. Sulphated material in macromolecular form was extracted from lymph nodes that had been labelled in vivo and was also found in the supernatant of lymph node cultures. The labelled material was not proteoglycan in nature. High endothelial cells apparently secrete a sulphated macromolecule but its relationship to the only known function of high-walled endothelium - the selective extraction of lymphocytes from the blood - remains to be clarified. - LYMPHOCYTES ACROSS MACROMOLECULE BY HIGH ENDOTHELIAL CELLS IN LYMPH NODES* The vascular endothelium lining the post-capillary venules of the lymph node paracortex was originally distinguished on morphological grounds (Thome", 1898; Schulze, 1925). Compared to the thin, flat endothelial cells lining small venules elsewhere, these specialized endothelial cells are increased in height as measured from the lumen of the vessel to the basement membrane, so that they have been described as high, plump or cuboidal. As reviewed by Andrews, Ford & Stoddart (1980), these high endothelial cells differ quantitatively from flat venular endothelium in many histochemical and ultrastructural characteristics. Gowans and his colleagues (Gowans & Knight, 1964; Marchesi & Gowans, 1964) discovered that the great majority of recirculating lymphocytes that enter lymph nodes (LN) from the blood do so by crossing the walls of high endothelial venules (HEV), arid recently the dynamic interaction between lymphocytes and HEV has been the subject of detailed ultrastructural study (Schoefl, 1972; Van Ewijk, Brons & Rozing, 1975; Anderson & Anderson, 1976). However, the relationship between the only known function of HEV - the selective extraction of lymphocytes from the blood - and their structural peculiarities remains totally obscure. It is particularly curious that an equally efficient mechanism for the selective extraction of lymphocytes from the blood is found in the marginal zone of the spleen, which lacks HEV. Early after the injection of sodium [ssS]sulphate into the footpad of rats a heavy autoradiographic localization of 3SS in the HEV of the popliteal LN was consistently observed, as has been reported in review papers (Andrews et al. 1980; Ford, Smith & Andrews, 1978). This signified that 35S had been incorporated into molecules that are rendered insoluble by fixation. The original reason for injecting [36S]sulphate was the suggestion that certain staining characteristics of HEV supported the presence of proteoglycan (Smith & Henon, 1959), although the validity of this conclusion has been seriously questioned on the basis of more detailed histochemical study (Ropke, Jorgensen & Claesson, 1972). The aims of the present paper are: first, to report studies on the time-course and precise site of [8SS]sulphate incorporation both in vivo and in vitro; and second, to describe the initial characterization of the sulphated molecule. This led to the conclusion that [36S]sulphate is incorporated into a macromolecule that is not a proteoglycan. Experiments intended to elucidate the relationship between the synthesis of this sulphated macromolecule, the structure of HEV and lymphocyte traffic across HEV will be reported in later papers. MATERIALS AND METHODS The rats used in this study were adult males or females of the highly inbred AO or PVG strains. Occasionally (AO x PVG) F t hybrids were used. Biochemicals were obtained from Sigma (London) Chemical Co., Poole, Dorset, U.K.; all other chemicals were of analytical grade where possible and were purchased from British Drug Houses, Poole, Dorset, U.K., unless otherwise stated. Methacrylate resin (JB-4) was obtained from Poh/science8 Inc., Warrington, Pennsylvania, U.S.A., whilst Epon 812 resin and glutaraldehyde were supplied by TAAB Laboratories Equipment, Reading, Berks., U.K. Dulbecco's balanced salt solution was purchased from Oxoid Ltd, Basingstoke, Hants., U.K. and sheep erythrocytes supplied by Tissue Culture Services Ltd, Slough, Berks., U.K. Carrier-free sodium [S5S]sulphate (SJS-2P) was obtained from The Radiochemical Centre, Amersham, Bucks, U.K. In vivo labelling of the high endothelial venule (HEV) The popliteal LN was studied because it receives a well-defined drainage from the hind footpad, which ensured that material injected into the footpads had rapid access to this LN (Drayson, Smith & Ford, 1981). Both hind footpads were injected with o-i ml of 10 % (v/v) washed sheep erythrocyte suspension to increase the size of the popliteal LN (Drayson et al. 1981) and the volume of HEV (Anderson, Anderson & Wylie, 197s). Five days later at the height of the proliferative response 1 -o (iCi g"1 body weight of sodium ["SJsulphate was injected into each footpad. The rats were killed by cervical dislocation at intervals from 15 min to 24 h after injection. Both popliteal LN were removed, cleaned of adherent fat and washed in Dulbecco's balanced salt solution A + B (DAB) before fixation by immersion in 3-0 % (v/v) glutaraldehyde in o-i M-sodium cacodylate buffer (pH 7-4) at 4 C for 2-3 h. Embedding for autoradiography Tissues to be examined by light microscopy were dehydrated through a graded ethanol series and embedded in methacrylate resin, according to the manufacturers, instructions (Polysciences Data Sheet, 123, May 1976). Sections were cut at 1-5 /im and prepared for autoradiography according to the method of Kopriwa & Leblond (1962) using Ilford G-5 emulsion. The sections were developed after 14 days using Amidol developer (Rogers, 1973) and stained with toluidine blue. After fixation, specimens for electron microscopy were thoroughly washed in o-i M-sodium cacodylate (pH 7-4) containing 3-0 mM-calcium chloride. Samples were post-fixed in o-i % (w/v) osmium tetroxide in o-i M-sodium cacodylate (pH 7-4) for 90 min at 4 C. All specimens were dehydrated in graded ethanol and embedded in Epon 812 resin before sectioning. Thin sections (100-150 nm) were cut on a Reichert OM U3 ultramicrotome and prepared for electron microscopic autoradiography by a loop technique (Caro & van Tubergen, 1962) using Ilford L-4 emulsion. After appropriate exposure, the preparations were developed with D-19 developer (Eastman Kodak Co.), double stained with uranyl acetate and lead citrate before examination in a Philips (...truncated)