LYSOSOMES IN RAT THORACIC DUCT LYMPHOCYTES FRACTIONATED BY ZONAL CENTRIFUGATION

A method of zonal centrifugation was developed which separates rat thoracic duct lymphocytes (TDL) mainly according to size. The validity of the fractionation method was supported by light microscope observations, Coulter Counter sizing, and in vivo and in vitro labeling of lymphocytes. The distributions of lysosomal acid hydrolases in TDL fractionated by zonal centrifugation are similar to the distribution obtained for the cells. This result indicates that the large lymphocyte is not the sole bearer of either lysosomes or the large amount of soluble cathepsin D found in homogenates of TDL. Both reside mainly in small lymphocytes. This point was clearly established by fractionating homogenates of purified small lymphocytes by means of differential centrifugation and isopycnic density gradient centrifugation.     

THE MIGRATION OF LYMPHOCYTES ACROSS SPECIALIZED VASCULAR ENDOTHELIUM

The chain of lymph-nodes in the rat mesentery was isolated and the preparation was perfused via cannulae in the superior mesenteric vessels. The perfusate consisted of serum to which labelled lymphocytes had usually been added. The entry of radioactively labelled lymphocytes from the blood vessels into the lymph-nodes was studied by scintillation counting and autoradiography. It was observed that: (1) In the perfused node labelled lymphocytes localized in and around post-capillary venules in the paracortex as they do early after i.v. injection. (2) The number of lymphocytes which entered the node was directly proportional to the concentration in the perfusate over the range studied. The proportion of cells retained in the node varied considerably around a mean of 11 % of lymphocytes reaching it. (3) The isolated lymph-node released few if any lymphocytes into the effluent (venous) perfusate. (4) Large lymphocytes migrated into isolated lymph-nodes slightly more readily than did small lymphocytes. (5) Unlike intact cells isolated lymphocyte membranes did not adhere to specialized vascular endothelium.     

THE MIGRATION OF LYMPHOCYTES ACROSS SPECIALIZED VASCULAR ENDOTHELIUM

Lymphocytes were exposed in vitro to either trypsin or neuraminidase. The ability of the treated cells to migrate into tissues was measured (a) by i.v. injection into intact recipients and (b) by vascular perfusion through an isolated lymph-node preparation. The localization of trypsinized cells in the lymph-nodes of recipients was deficient when compared to untreated lymphocytes and there was a surplus of trypsinized cells in the blood. Trypsinized cells migrated into the isolated nodes in reduced numbers. By contrast, neuraminidase treated lymphocytes were markedly deficient in the blood of recipients early after injection; their localization in the spleen and lymph-nodes was also deficient but they were in surplus in the liver. Moreover they migrated into the isolated nodes in slightly increased numbers. By 24 hr after injection the perturbed localization pattern produced by either enzyme was partly restored to normal. In conclusion, trypsin interfered with the capacity of lymphocytes to migrate into lymph-nodes but neuraminidase did not; the latter promoted the hepatic sequestration of cells and the reduced localization in the blood and tissues was a consequence of this. The hypothesis that lymphocytes adhere to specialized endothelia in lymph-nodes because of specific glycoside sequences on their surface lacks experimental support.     

SOME OBSERVATIONS ON THE ESTIMATION OF TRYPTAMINE IN TISSUES

The method of Hess and Udenfriend (1959) for the estimation of tryptamine in tissues, is demonstrated to allow the carry over of tryptophan to the final extract, resulting in falsely high values for the amine. A modification of the original method is described, incorporating ion-exchange chromatography, which gives complete separation of these components. Using this method, tryptamine is found neither in the brain of normal guinea pigs, nor in animals treated with l -tryptophan. It is found only when l -tryptophan is administered following pretreatment with an amine oxidase inhibitor.     

MIGRATION OF SMALL LYMPHOCYTES IN ADULT MICE DEMONSTRATED BY PARABIOSIS

Parabiotic BALB/C mice were used to study the traffic of small lymphocytes in immunological mature but unchallenged mice. By giving ³H-thymidine (³H-TdR) injections to only one member (A) of a pair by preventing the escape of the radioactive isotope to the other member (B), the kinetics of newly-formed cells was followed. Less than 10% labelled small lymphocytes were found in the peripheral lymphoid tissues of both A and B members, while the thymusses and bone marrows of A members showed labelling percentages up to 70% in this period. Hardly any labelled cells gained entrance into the thymus while a detectable number was found in the bone marrows of B members. Results from pairs set up to follow migration of long-lived lymphocytes revealed that labelled cells detected 4–5 weeks after injections were equilibrated between the peripheral tissues and the bone marrows of the partners. Very few labelled cells were seen in the thymic medulla and none were observed in the thymic cortex, germinal centres or medullary cords of lymph nodes from any B member. It was concluded that short-lived small lymphocytes are formed primarily in the thymus and bone marrow and the migration of these cells is limited in adult animals. Furthermore, the vast majority of long-lived small lymphocytes are freely recirculating, and these cells gain entrance to and are normal residents in the bone marrow.     

THE NATURE OF THE FACTORS WHICH DETERMINE THE SEQUENCE OF GROWTH-CYCLES AND ITS RELATIONSHIP TO THE DIFFERENTIATION OF TISSUES

1. It has previously been shown by the author and many others that growth, in animals and plants, is an autocatalysed process. In animals it is usual to find that growth occurs in several superimposed autocatalytic cycles. In many cases, in plants and animals, especially if the cycle is one which occupies a large proportion of the growing period, it is found that the velocity-constant of the autocatalysed monomolecular formula falls off as growth proceeds, at first rapidly and later more slowly. 2. It has previously been shown by the author that the fall of the velocity-constant of growth, in the white mouse, is directly proportional to the fall of the nucleo-cytoplasmic ratio, determined by the chemical method of Le Breton and Schaeffer. If we assume this relationship to be generally applicable to the growth of animals and plants, then the following additional conclusions may be deduced, without calling in the aid of any other assumption:— 3. The increase of cytoplasm in any given cycle of growth is proportional to the concurrent increase of nuclear material. 4. The growth of cytoplasm takes place in accordance with a monomolecular formula in which the velocity-constant varies directly as the mass of the nucleus. If we superadd to these facts and deductions the hypothesis that each growth-cycle represents the growth of a separate group of cells within the animal, then the additional conclusions follow:— 5. That the cells which participate in the growth composing any cycle have initially lower nucleo-cytoplasmic ratios than the cells which participated in the preceding cycles. 6. That cells of large nucleo-cytoplasmic ratios in a multicellular animal inhibit the growth of cells which possess smaller ratios. 7. These conclusions collectively imply that the nucleus plays a predominant role in determining the development of the cell in which it resides.