L Y S O S O M E S I N L Y M P H O I D T I S S U E III. Influence of Various Treatments of the Animals on the Distribution of Acid Hydrolases

The density-distribution patterns of various enzymes and of labeled materials have been determined by isopycnic centrifugation in a sucrose-0.2 M KC1 gradient on homogenates of lymphoid tissues from rats injected with Triton WR-1339, 14C-labeled dextran, 51Cr-labeled erythrocytes, and cortisol. The results confirm and extend the conclusion, derived from previous investigations on normal animals, that the lysosomes of lymphoid tissues form two and possibly three, distinct populations. The evidence indicates that the L19 population belongs to macrophages and the Lx5 group to lymphocytes. The L30 population appears to be associated with a special type of phagocyte with a high capacity for dextran storage. All three populations seem to contribute to the activities found in soluble form in homogenates of normal lymphoid tissues. I N T R O D U C T I O N One of the main functions of lysosomes is concerned with the storage and digestion of exogenous materials taken up intracellularly by an endocytic process (for a review, see reference 12). In the present work, advantage has been taken of this phenomenon to characterize further the various lysosomal populations tentatively identified in rat lymphoid tissue as LI~, L19, and L30 (4,5). The following treatments were selected for application to rats, from which the spleen was subsequently removed and fractionated by isopycnic centrifugation in a sucrose-0.2 M KC1 gradient: 1. Injection of Tri ton WR-1339, a substance shown by Watt iaux and coworkers (39-41) to accumulate in liver and kidney lysosomes and to cause a marked decrease in the equil ibrium density of these particles in a sucrose gradient. 2. Injection of 14C-labeled dextran, a polysaccharide known from morphological observations to become concentrated in the lysosomes of liver (8,10) and of some spleen cells (9), and found to increase the equil ibrium density of hepatic lysosomes in a sucrose gradient (2). 3. Injection of 51Cr-labeled erythrocytes, as an easily detectable substrate for the well known erythrophagocytic function of spleen. In a final series of experiments, the lympholytic effect of cortisol (13) was used to modify the cellular composition of spleen, thymus, and lymph nodes, and the changes incurred by the lysosomes of these tissues were investigated by densitygradient isopycnic centrifugation. M A T E R I A L S A N D M E T H O D S Triton WR-1339 (Rohm & Haas Co., Philadelphia, Pa.), dissolved in saline, was injected at the dosage of 75 mg per 100 g rat into one of the lateral tail veins under light ether anesthesia. Dextran was injected similarly at a dosage of 40349 on O cber 0, 2017 jcb.rress.org D ow nladed fom 60 nag per 100 g rat. T h e labeled mater ia l given consisted of a 5:3 mix ture of cold dextran-500 (Pharmacia , Stockholm, Sweden) and of the same polysaccharide tagged per ipheral ly by incubat ion in the presence of uni formly labeled sucrose-t4C and of dex t ran sucrase extracted f rom Leuconostoc mesenteroides (prepared by Dr. P. Jacques) . This mater ia l was injec ted either as such, or with an equal a m o u n t of dextran-10. Erythrocytes were obta ined f rom rat or mouse blood collected into S t rumia (38) mix ture con ta in ing 0 .1% of heparin . T h e ra t blood was kept overnight at 2°C in this mixture , wi th addi t ion of formaldehyde to a final concentra t ion of 0.5%. It was then centrifuged at 600 g for 10 rain and the sedimented cells were resuspended in S t rumia mix tu re and incubated for 5 m i n at 37°C wi th 10 #curies of Na-51CrO4 (Rachromate 51, Abbot t Laboratories, Chicago, Ill.) (26). The ceils were washed free f rom excess ch romate by five to six washings wi th S t rumia mix tu re and injec ted intravenously in an a m o u n t corresponding to 0.2-0.3 ml of blood per 100 g rat. T h e mouse blood was t reated in the same way, except tha t the incubat ion with formaldehyde was omit ted. I t has been shown tha t in this labeling procedure 51Cr binds to the be ta cha in of hemoglobin (31). Cortisol (Sigma Chemica l Co., St. Louis, Mo.) was injected subcutaneous ly twice dai ly at the dosage of 15 m g per 100 g rat. T h e lympho id tissues of the t reated an imals were fract ionated by isopycnic centr i fugat ion in a sucrose0.2 M KC1 gradient and analyzed, as described in the 8O0 r,~--, IN-AcetylI I B-Glucosominidose ! i..