ABSTRACT
High endothelial cells lining the post capillary venules in the paracortical areas of rat lymph nodes were found by autoradiography to incorporate [35S]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 [35S]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.
Subject(s)
Lymph Nodes/blood supply , Lymphocytes/physiology , Sulfates/metabolism , Veins/metabolism , Venules/metabolism , Animals , Autoradiography , Cell Movement , Endothelium/metabolism , Endothelium/ultrastructure , Female , In Vitro Techniques , Lymph Nodes/ultrastructure , Macromolecular Substances , Male , Microscopy, Electron , Molecular Weight , Rats , Rats, Inbred Strains , Venules/ultrastructureABSTRACT
Collagenolytic cathepsin activity was detected in lysed rabbit peritoneal polymorphonuclear leucocytes. The pH optimum was around 3, and activity was greatly enhanced by the presence of cysteine and EDTA. Digestion of polymeric collagen resulted in the release of alpha, beta, and gamma-chains. Collagenolytic cathepsin activity was associated mainly with the granule fraction isolated from homogenates by differential centrifugation. The granule fraction was further fractionated by isopycnic density-gradient centrifugation, and the collagenolytic cathepsin activity was shown to be associated with the azurophil and tertiary granules, both lysosome-like organelles.
Subject(s)
Cathepsins/metabolism , Neutrophils/enzymology , Animals , Centrifugation, Density Gradient , Collagen/metabolism , Cysteine/pharmacology , Edetic Acid/pharmacology , Hydrogen-Ion Concentration , In Vitro Techniques , Rabbits , Subcellular Fractions/enzymologyABSTRACT
Human polymorphonuclear leucocytes were obtained from the synovial fluids of patients with inflamed knee joints suffering either from Reiter's syndrome or from rheumatoid arthritis. The polymorphonuclear leucocytes were collected by gentle centrifugation followed by disruption and their subcellular fractionation by centrifugation in 0.34 M sucrose to provide a granule fraction and a post-granule supernatant fraction. 0.5 M KCl extraction of the granule fraction yielded neutral protease activity, similar to trypsin, when assayed against fluorescein-labelled polymeric collagen fibrils. The post-granule supernatant fraction contained an inhibitor towards the neutral protease and trypsin. The inhibition of the neutral protease was found to be time-dependent, this inhibition being released after 1.5-2 h. In contrast, the inhibition of trypsin was irreversible and this property was used to devise an assay procedure for the inhibitor.
