Medium Lymphocytes Research Articles

Free-flow electrophoretic separation of lymphocytes. Two thoracic duct lymphocyte subpopulations studied after prolonged cannulation and immunization.

AbstractTwo different populations of small thoracic duct lymphocytes were separated by electrophoresis and characterized by electron microscopy. The high electrophoretic mobility region (HMR) consisted of small lymphocytes only and contained 75 % of the cells. After thoracic duct drainage or antigenic stimulation the morphological characteristics of the cells in the HMR remained unchanged. In contrast, the cells in the low electrophoretic mobility region (LMR) increased in quantity and showed morphological alterations after prolonged duct drainage or antigenic stimulation. Prior to stimulation small and medium lymphocytes were found in the LMR. After stimulation, blast‐like cells, immature plasma cells and “activated” lymphocytes, in addition to increased numbers of small lymphocytes, were detected.

Immunoglobulin spots on the surface of rabbit lymphocytes.

Small and medium lymphocytes from the peripheral blood and lymphoid tissues of the rabbit react in suspension with antibodies directed against different immunoglobulin determinants. Through immunofluorescence, it was possible to show that numerous discrete spots on the surface of the positive lymphocytes carry immunoglobulin molecules. The positive lymphocytes are about one-half of all lymphocytes in the different preparations; thymus lymphocytes are all negative. With antisera specific for rabbit IgM as well as with antisera directed against allotypic determinants specific for IgM or IgG, it was possible to show that about nine-tenths of the immunoglobulin-positive lymphocytes carry IgM molecules on their surface. With antisera directed against a- and b-locus determinants, it was also possible to demonstrate that both heavy and light chains were present in the surface immunoglobulins. Furthermore, in animals which were heterozygous at the a or the b locus, it was found that each lymphocyte had immunoglobulins synthesized under the influence of only one of two alleles. A very small proportion of lymphocytes could be shown to have a specific surface reaction with one antigen (horse ferritin); the proportion of these cells increased very much after immunization.

H3‐leucine incorporation in antigenically stimulated rat popliteal lymph node cells

AbstractThe effect of antigenic stimulation with pertussis and sheep red blood cell antigens was studied in the rat popliteal lymph nodes. H3‐leucine was used to assess protein metabolism in the various cell types of the reactive lymph nodes following both primary and secondary stimulation with these antigens. Radioautographs of tissue smears were used to make quantitative and qualitative assessments of the extent of intracellular protein synthesis. In addition, changes in the cellular populations of various lymphoid cell lines were determined utilizing differential cell counts and total cells present in the reacting nodes. These data were used principally to assess the role of the lymphocytic cells in the ontogeny of the immune response.Large and medium lymphocytes responded to antigenic stimuli by increasing intracellular protein synthesis. This newly synthesized protein most likely reflected the requirements of large and medium lymphocytes for growth, division and the production of immunoglobulins. The small lymphocytes, as a group, showed only minimal changes in intracellular protein synthesis following antigenic stimulation.The responses by large and medium lymphocytes and plasma cells were qualitatively similar following primary and secondary stimuli. The differences in these responses appeared to be reflected by the number of cells involved and by the temporal sequence in which the cells appeared.

Techniques for the long-term collection of lymph from the unanaesthetized foetal lamb in utero.

SummarySurgical techniques are described for establishing chronic fistulae in the thoracic, intestinal, lumbar and popliteal lymph ducts of foetal lambs in utero. These fistulae have remained patent and enabled lymph to be collected for periods of up to 5 days. The Iambs have been born spontaneously or delivered by Caesarean section with their fistulae intact and still flowing.Lymph flow rates were 9–18 ml./hour from the thoracic duct, 2.8–8.6 ml. /hour from the intestinal duct, 2.8–9.2 ml./hour from the lumbar trunk and 0.6 ml./hour from the popliteal duct.The protein concentration of foetal thoracic duct lymph was 2.30 g./100 ml. immediately after cannulation, the intestinal lymph 2.37 g./100 ml. and the lumbar lymph 2.12 g./100 ml.The cells of foetal lymph were almost all small and medium lymphocytes and only an occasional cell was found in mitosis. Drainage of the thoracic duct lymph for 36 hours drastically reduced the number of lymphocytes in the lymph, suggesting that, as in the adult sheep, most of the lymphocytes in foetal lymph are recirculating cells.

Lymphoid cellular responses in the blood after immunization in man.

A combined morphological and metabolic study has been made of the lymphoid cells in the blood during the immune response in man. Similar changes were observed in both primary and secondary responses to a number of different microbial antigens. The cellular response involved an increase in numbers of three types of cell; hyperbasophilic medium lymphocytes, plasma cells, and large lymphoid cells. The large lymphoid cells were about 20 micro in diameter with large nuclei, prominent nucleoli, and an intensely basophilic cytoplasm with numerous polyribosomes. About 30% of these cells were in the DNA synthetic phase of cell growth. Electron microscopy has shown that many of the basophilic medium-sized cells have sufficient well-organized endoplasmic reticulum to be included in the plasma cell series. The hyperbasophilic cells labeled more heavily with tritiated uridine and tritiated leucine than the normal small and medium lymphocytes from the peripheral blood of patients not under antigenic stimulation. The evidence in this paper supports the argument that the atypical mononuclear cells first described by Türk and others in the blood of patients with infections are immunoblasts, plasma cells, and other reactive lymphoid cells representing a circulating population of lymphoid cells derived from lymphoid tissue responding to antigenic stimulation. The presence of such cells may be a valuable indication that an immunological reaction is in progress when direct proof is lacking.

Cell Population Analysis of Radiation Damage and Recovery of Thymus and Lymph Nodes in Mice

C57BL/6 mice received whole-body x-irradiation, and differential cell counts were made on cellular suspensions of the lymphatic tissues at regular intervals. A two-phase pattern of thymic lymphopoiesis was demonstrated: The first phase of blast cell proliferation lasted for three to four generations and was followed by a period of differentiation to small lymphocytes. The difference in the time of repopulation between the thymus and the lymph nodes was ascribed to the difference in the absolute number and the doubling time of primitive cells. The doubling time of blast cells was 12 hours in thymus and 20 hours in lymph nodes. The relation between dose and the number of surviving cells at 24 hours after irradiation revealed the existence of two classes of lymphocytes with different radiosensitivity. The $D_{37}$ was 145 R and 425 R for small lymphocytes and 57 R and 520 R for medium and large lymphocytes in thymus. The $D_{37}$ values for small and larger lymphocyt...

Studies on the Blood Morphology of Three Estuarine Cyprinodontiform Fishes

Specimens of Fundulus heteroditus, Fundulus majalis, and Cyprinodon variegatus were bled in the field at the site of capture. Blood was obtained during all seasons and in the laboratory at intervals over a 30-day period from specimens held in two types of holding aquaria. The circulating blood elements of these three estuarine species consist of erythrocytes, small and medium lymphocytes, eosinophils, and thrombocytes. The observations were made using both stained and fresh blood preparations.Variations occurred seasonally in the morphology of erythrocytes, eosinophils, and thrombocytes. Cytoplasmic inclusions were visible in erythrocytes prior to, and at the beginning of, the new breeding season. There were changes in the refringent properties of thrombocytic and eosinophilic granules, and cyclic variations in the number of eosinophilic granules. Seasonal variations occurred in the ratio of immature to mature thrombocytes, but there was little overall variation in the ratios of leucocytic cell types. The thrombocytes were characterized by thrombocytic granulation and nuclear indentations. Thrombocytes were capable of altering their form; mature cells formed thrombocytic networks.The concentration of eosinophils gradually increased to 18% above the usual level in Fundulus heteroditus held 30 days in 20-liter circular aquaria. The eosinophil level remained at the usual 2% in specimens held in 189-liter aquaria for the same time period. Values for specimens captured in a natural environment were also 2%. It is therefore plausible to relate the rise in eosinophils with an environmental stress associated with the circular holding containers.

Studies on the anti-inflammatory action of 6-mercaptopurine.

The mechanism of action of 6-mercaptopurine (6-MP) on an egg albumin-induced inflammatory lesion in the skin has been studied in rabbits treated with 6-MP in a daily dosage of 18 mg/kg. Relative to control animals, significant decreases in the numbers of large lymphocytes and monocytes in the blood were observed in the 6-MP-treated animals by the 9th day of treatment, without significant decrease in the numbers of polymorphonuclear leukocytes and small and medium lymphocytes. Concurrently, a significant decrease was also seen in the percentage of tissue mononuclear cells in the inflammatory skin lesion. There was a highly significant correlation between the numbers of monocytes in the blood and the per cent of mononuclear cells in the lesion. A mean of 52% of the mononuclear cells in the tissue lesion phagocytosed carbon offering further evidence that the major cell involved was the blood monocyte. In vitro incorporation of (3)H-Tdr by blood mononuclear cells was significantly reduced in the 6-MP-treated animals as determined by scintillation counting and radioautography. The large lymphocyte was the predominant cell type which was labeled in vitro. Small lymphocytes and monocytes were rarely labeled. The data obtained suggest that the anti-inflammatory effect of 6-MP, reflected in these experiments by a decrease in mononuclear cells in a tissue lesion, results from suppression of a bone marrow response to local inflammation, affecting principally proliferating precursors of blood monocytes and large lymphocytes. The possible importance of this action of 6-MP in the treatment of inflammatory and immunologically mediated disease is discussed.

The in vivo induction of mouse lymphocyte transformation by phytohemagglutinin

Summary The effects of the intravenous administration of phytohemagglutinin-M (PHA-M) and phytohemagglutinin-P (PHA-P) on the mesenteric lymph node, spleen, bone marrow, thymus and peripheral blood of the mouse have been studied. Qualitative changes similar to that observed when PHA is used in vitro with lymphocytes were found. There was a marked shift in the differential of the mesenteric node and peripheral blood from small lymphocytes to medium and large lymphocytes, many of which resemble the blast-like cells seen in tissue culture. Both the shift in differential and the increase in proportion of large and medium cells in deoxyribonucleic acid synthesis in the mesenteric node were maximal at 3 days after the administration of PHA. An increase in the weight of the spleen and the proportion of cells in mitosis and in DNA synthesis was also maximum at 3 days, whereas in the bone marrow the maximum effect occurred only 1 day after PHA. No increase in weight of the thymus, shift in the differential or increase in the proportion of cells in DNA synthesis or mitosis was found with PHA-M, but PHA-P did have a slight effect on all three of these parameters. As a result of its erythro- and leukoagglutinating properties, PHA-M caused a decreased hematocrit, red blood cell count and peripheral platelet count with a compensatory increase in the absolute number of megakaryocytes of the bone marrow 3 days after its administration.

Origin of Plasma Cells in the Thymus

The origin of plasma cells in the thymus as well as in the lymphoid organs and sites of inflammation is still being discussed. Moore and Schoenberg believed that in experimentally induced inflammation they developed from both small and medium lymphocytes. Nossal and Keuning concluded that plasma cells originated from a primitive lymphocyte in lymph nodes. In the thymus they are absent during the first weeks of age, but from the sixth week on they are easily recognized even under the light microscope, and their number appears to increase during the following weeks.In the present work we examined the corticomedullary junction area of the thymus in albino rats at progressive ages, starting at an hour after birth, and then at weekly intervals until the twelfth week. The appearance of the thymus during the first weeks is rather simple; it consists mainly of islets of undifferentiated epithelial reticular cells, surrounded by small and medium sized lymphocytes.

Effects of Continuous Low-Level Gamma Irradiation on Circulating and Peritoneal Mononuclear Leukocytes of Mice

Abstract : LAF1 mice were exposed continuously to Co60 gamma radiations at a dose rate of 1.4 rads per hour. The number of lymphocytes in the circulating blood fell sharply during the first week of exposure (190 rads) and decreased thereafter at a very gradual but statistically significant rate for the duration of the experiment (15 weeks, 3450 rads). The disappearance of small lymphocytes (6 microns in diameter) from the peritoneal cavity was also more rapid during the first week of irradiation than during subsequent weeks. Medium peritoneal lymphocytes (8-10 microns in diameter) and peritoneal macrophages disappeared at constant rates over the entire observation period. After the first week of exposure, the disappearance rates of small and medium peritoneal lymphocytes were identical. This rate was greater than that for peritoneal macrophages and that for circulating lymphocytes. Based on the fraction of cells surviving any given exposure, the mononuclear leucocytes may be arranged in the following order of decreasing sensitivity to continuous low dose rate gamma irradiation: circulating lymphocytes, small peritoneal lymphocytes, medium peritoneal lymphocytes, peritonela macrophages. This order is the same as that after acute exposure to X rays. (Author)

Lymphocytes and Transplantation Immunity

This chapter discusses different aspects of transplantation immunity and presents complete review of the lymphocyte and its role in different types of immunological reactions in experimental animals and in in vitro systems. The lymphoid tissues of a normal mammal constitute about 1% of its body weight and the predominant cellular component is the “small” lymphocyte. Lymphocytes serve as trephocytes and stem cells and even regulators of tissue growth and size. On the basis of their morphology and size, lymphocytes are subdivided into “large,” “medium,” and “small” varieties. The majority of the lymphocytes present within the lymphoid tissues as well as in the blood and lymph are of the “small” variety. The large and medium lymphocyte categories represent a much more heterogeneous population of cells, some of which possess a strongly basophilic cytoplasm. Electron microscope studies of the ultrastructure of these cells reveals two types, both distinguishable from small lymphocytes on the basis of their cytoplasmic organelles and their more abundant cytoplasm. Among the characteristics of small lymphocytes, their ubiquity, mobility, and longevity are desirable attributes of any cell type the role of which is to participate in the recognition, effector, and memory aspects of immunological phenomena. The recirculating small lymphocytes, rather than the sessile lymphoid cell population, play an important role in the primary response of rats to skin homografts.

Effects of Total-Body X-Irradiation on Peritoneal Cells of Mice

Abstract : Peritoneal cells of normal and irradiated LAF1 mice which had not been pretreated with peritoneal irritants were studied by phase-contrast microscopy and in stained smears. The average number of leucocytes recovered per normal mouse was 7.4 million. Approximately 95% of the cells were mononuclear; of these, macrophages comprised 29% (2.2 million), small and medium lymphocytes 71% (as determined by phase-contrast). One day after whole-body exposure to 690 R (LD30/30) or 720 R (LD50/30) the mean leucocyte count was 3.4 million, macrophages accounted for 65% (2.2 million) of the cells. From 3 to 21 days postirradiation, cell counts gradually decreased further to 1.5 million. Macrophages decreased at the same rate; they constututed approximately 79% of the cells during this period. Thereafter, the number of leucocytes increased and reached normal levels by the 12th week postirradiation. When suspensions of viable Escherichia coli or Staphylococcus aureus were injected 30 minutes before sacrifice, the macrophages of both normal and irradiated mice contained large numbers of microorganisms. Thirty minutes after injection of bacteria or E. coli lipopolysacchride (10 micrograms) into normal mice, the number of cells in peritoneal washings was not altered. However, after injection of these agents into irradiated mice, total cell counts were significantly lower than in uninjected or diluent-injected mice.

The effect of phytohemagglutinin on rat thymus cells in vitro.

AbstractCells obtained from the thymus glands of five strains of rats, ranging in age from five hours to five months, were cultured in the presence of phytohemagglutinin according to the method of Rieke and Schwarz ('64). In these cultures, the large and medium lymphocytes in the inoculum gave rise by mitosis to small lymphocytes. Under the influence of PHA, small lymphocytes were transformed into enlarged cells which in turn acted as precursors of small lymphocytes. However, not all small cells in the cultures nor thymic lymphocytes from newborn rats responded to this mitogenic drug. A progressive increase in responsiveness was detected as donors increased from five hours to 32 days of age.

A radioautographic investigation of the identity of phytohaemagglutinin responsive cells in the lymphoid tissues of the rat

Summary In phytohaemagglutinin stimulated cultures of rat lymph node, spleen, thymus, blood and thoracic duct lymph studied radioautographically: o 1. Large and medium lymphocytes do not participate in the cellular transformation. 2. Small lymphocytes are stimulated to growth and replication of DNA. 3. Both “long-lived” and “short-lived” small lymphocytes respond to PHA stimulation. It is difficult to reconcile this latter finding with the view that the mitogenic action of PHA is the result of a specific immunologic type of reaction.

Elaboration of a Model for the Formation of Lymphocytes in the Thymic Cortex of Young Adult Rats

Abstract In the hope of clarifying the sequence of cells in thymus, mitotic figures were classified into four types and counted in the cortex and medulla of thymus of young adult rats. The four types were designated as reticular cell mitoses (characterized by an unstained cytoplasm with a very large mitotic figure), and lymphocyte mitoses (characterized by a basophilic cytoplasm with a mitotic figure, which may be large, 5.6-8.0 µ, medium sized, 4.5-5.6 µ, or small, 3.5-4.5 µ). The relative mitotic counts of the four cell types in thymic cortex were 3, 46, 172 and 240 respectively. Analysis of these counts, assuming equal mitotic duration and orderly sequence of the cells led to two sets of conclusions. (1) Large, light cells, the divisions of which are referred to as reticular cell mitoses, would produce large lymphocytes. These cells would proliferate to give rise to medium lymphocytes, which in turn would proliferate to give rise to small lymphocytes. (2) There would be 2-4, most probably 4, successive generations of large lymphocytes, followed by 2 successive generations of medium lymphocytes, which would in turn be followed by 1 generation of mitosable small lymphocytes, probably giving rise to another generation whose cells would not divide, at least in thymic cortex. A model of lymphocytopoiesis for the cortex of thymus is proposed on the basis of these conclusions.

The Blood Picture of the Goat: IV.—Changes in Coagulation Times, Platelet Counts and Leucocyte Numbers, Associated with Age

SUMMARY Coagulation times, platelet counts and absolute and differential leucocyte counts were estimated in goats from the day of birth to the age of 3 years and regression lines were calculated from the data. The results are detailed in Tables and Figures. Blood coagulation times fell from an average of 4·25 min. near birth to 2·9 at 68 hr. They then rose slowly to reach 4·49 at Month 30. Platelet counts averaged 116,130 per mm.3 over the first 1 days and then began to fall until Mouth 17 or 18 when they reached their minimum of 28,000. They then rose to 62,500 at Mouth 23. Total leucocyte counts averaged 8,371 per mm.3 over the first 14 days; the average then rose to reach 19,180 at Week 16 and subsequently fell to 10,931 at Month 30. In the differential count monocytes remained at an average of 2·54%, eosinophils at 0·984 and basophils at 0·278% in spite of the changes in total leucocyte count. Immature neutrophils rose from an average of 0·42% to 2·5% on the 4th day and then fell to 1% in Week 2. No young forms were seen after Month 4 and baud forms were rare after Month 14. Absolute counts of neutrophils and lymphocytes showed a quick rise in lymphocytes from 2,930 per mm.3 over the first 16 hr: to 14,661 at Mouth 4, followed by a slow fall to 3,890 at Month 39. Neutrophil changes were less marked and the average remained between 3,000 and 4,800 per mm.3 Medium lymphocytes with nuclei containing bodies resembling nucleoli are discussed.

EFFECT OF THYMECTOMY ON HEMATOPOIETIC ORGANS OF THE OPOSSUM "EMBRYO".

SummaryOpossums were thymectomized when lymphatic tissue had developed in some lymph nodes but not in the spleen. They were sacrificed 2 weeks later. Lymph nodes were grossly deficient in small and medium lymphocytes and lymphatic tissue did not appear in the spleen. Myeloid tissue not only persisted but was found greatly increased in amount at a time when it would have normally decreased. Maturation and proliferation of this tissue was abnormal, being characterized by maturation arrest and ineffective erythropoiesis. These findings indicate that the thymus influences the origin of lymphatic tissue, at least in the spleen, maintenance of lymphatic tissue in lymph nodes, and suppression of myeloid tissue in lymph nodes and spleen.

Cellular localization of rapid RNA and protein synthesis induced in presensitized lymphocytes by a cationic protein from brain

Results of previous biochemical experiments indicated that lymph node cells, presensitized to a purified cationic protein from guinea pig brain, were induced to undergo rapid protein synthesis when incubated in the presence of the basic protein. An incubation period of 1 hr was required before the increase in protein synthesis could be observed. In the autoradiographic studies here reported, the nuclei of presensitized cells which appear to be medium lymphocytes, gave evidence of rapid RNA synthesis (possibly messenger RNA) during the initial hour of incubation with the cationic protein. During the second hour of incubation the high rate of protein synthesis was observed in both small and medium lymphocytes. With the aid of fluorescent antigen it was demonstrated that the medium sized lymphocytes contain a material that specifically binds the cationic protein. The role of the medium lymphocyte in the induction of hypersensitive reactions is discussed.

The fine structure of lymphoid tissue

The fine structure of the lymphoid tissue is described. Six cell types can be identified and described with reasonable accuracy: reticular cells, macrophages, littoral cells, stem cells, lymphocytes, and plasma cells. The reticular cell resembles the fibroblast in having a pale elongated nucleus, branching cytoplasmic processes, a varying amount of ergastoplasm, and often an organelle-free portion at the cell border (ectoplasm), with delicate filaments. Marcophages may resemble reticular cells in shape or may be more rounded. The pale nucleus is elongated or kidney-shaped and contains a small nucleolus. The nucleus is often eccentric. In the cytoplasm there may be varying amounts of phagocytosed material and lysosomes. There are usually many smooth-surfaced vesicles, and the ergastoplasm is often confined to a more or less circumscribed area. Characteristic are the numerous fingerlike processes at the cell border, which may interdigitate with those of neighboring cells. Littoral cells resemble reticular cells and ordinary vascular endothelial cells. They show little or no phagocytosis under normal conditions, but may pick up certain substances, e.g., carbon, just like ordinary macrophages. The lymphocyte nucleus is more or less rounded, most dense in the small lymphocyte, and least densc in the lymphoblast. Lymphoblasts and medium lymphocytes have nucleoli. The nucleo-cytoplasmic ration of small lymphocytes is the greatest of any cell. The cytoplasm is pale and contains a moderatc number of free ribonucleoprotein granules (ribosomes), well-formed mitochondria, and a small Golgi region. In addition, particularly in the medium lymphocytes, the cytoplasm has bodies which probably correspond to the Gall bodies of light microscopy. Plasma cells are characterized by a well-developed ergastoplasm that fills the cytoplasm to its margins, a large and complex Golgi apparatus, fingerlike cytoplasmic processes, and a densc eccentric nucleus. Immature plasma cells have a prominent nucleolus. The variants of the reticular cell are discussed, as is the evidence for and against the existence of a primitive multipotent reticular cell. A cell referred to as a “stem cell” is described. It is found occasionally in normal lymphoid tissue but frequently soon after antigenic stimulation. Its significance and the origin of lymphocytes and plasma cells are discussed. Extracellular elements in the spleen consist of reticular fibers and basement membranes. The latter support the littoral cells of the sinusoid. The reticular fibers of light microscopy are made up of delicate collagen fibrils, an occasional elastic fiber, and an interfibrillar ground substance.