Lymphopoiesis in the rat. II. The effect of thymectomy

Lymphopoiesis with respect to recirculating and non-recirculating small lymphocytes was measured simultaneously in rats thymectomized as adults. Removal of the thymus at four to five weeks of age had a profound inhibitory effect upon the production of recirculating cells, whereas the formation of non-recirculating lymphocytes was only slightly depressed. Thymectomy had approximately the same impact of lymphopoiesis as thymectomy and exposure of the animal to a large dose of whole body X- and γ-irradiation. The latter finding, and the failure of a thoracic duct cell transfusion to augment lymphocyte production, accord with the view that the thymus is the principle intermediate source of recirculating small lymphocytes in the normal, unstimulated animal.     

Virus-induced alterations of lymphoid tissue. IV. The effect of Newcastle disease virus on the fate of transfused thoracic duct lymphocytes

⁵¹Cr-labeled thoracic duct lymphocytes were briefly incubated at 4 °C with Newcastle disease virus (NDV) and then infused into syngeneic rats. Virus diverted the homing of many donor cells from lymph nodes and spleen to the liver. Evidence was obtained suggesting that some NDV-treated lymphocytes initially trapped in the liver subsequently migrated into the lymph nodes. The results imply that NDV transiently interrupts the normal route of lymphocyte migration. Alterations in lymphocyte distribution were mediated by attachment of virus to the cell surface and were the same as those induced by incubating lymphocytes with V. cholera neuraminidase before infusion. It appears that reactions involving 2–3′ and/or2–8′ linked sialyl residues on the surface of recirculating lymphocytes can markedly affect their distribution in the body.     

Migration of recently divided B and T lymphocytes to peritoneum and lung

It is recognized that a population of newly divided (or young) cells migrate preferentially to inflamed foci. It has been shown that a large proportion of lymphocytes residing in the bronchoalveolar airspaces of rat are recently divided cells and that blood may be an important source of these cells. To further delineate how blood may contribute to lymphocyte subpopulations in inflamed peritoneum and lung, a comparison of the capacity of recently divided T and B cells to migrate from blood to inflamed peritoneum and lung was made. To label young lymphocytes, DA strain donor rats were given Initiated thymidine by vein in vivo for 7 days. After thoracic duct drainage, the following labeled cell populations were adoptively transferred by vein into syngeneic recipients: (i) unseparated thoracic duct lymphocytes (TDL), (ii) enriched T cells (>90%) or B cells (>80%) recovered after passage of TDL through nylon columns, and (iii) thoracic duct lymphocytes (> 99% B cells) obtained from “B rats” that were prepared by X irradiation, thymectomy, and bone marrow reconstitution. T and B cells were identified by specific heterologous antisera. The percentage recovery of labeled lymphocytes in the recipients with inflamed peritoneum or lung aspirates was determined from cell counts and autoradiographs. The studies indicated that (a) both labeled T and B cells migrated to inflamed peritoneum and lung; (b) labeled B cells migrated to peritoneum and lung better than did labeled TDL or T cells; and (c) labeled lymphocytes did not migrate to unstimulated peritoneum. The enhanced migration of newly divided B lymphocytes to inflamed peritoneum and normal lung (a site that is likely under chronic antigenic stimulation) was unexpected, but may provide additional information on the relative contribution of these subpopulations in the immune inflammatory response.     

The circulating life span, immunocompetence and 3H-uridine uptake of small lymphocytes from thymus-grafted, neonatally thymectomized rats.

By 7 weeks post-grafting, the number of small lymphocytes in the thoracic duct lymph (TDL) and blood of the thymus-grafted neonatally thymectomized adult rats had increased to 60% of the number of cells in sham controls, or 2-1/2 times thymectomized control values. This increasing consisted almost exclusively of long-lived, recirculating small lymphocytes and corresponded to a 60% recovery of cellular immunocompetence as measured by the mixed lymphocyte reaction (MLR). Associated with the return of cellular immunocompetence was an increased incorporation of 3H-uridine by the small lymphocytes. Cells from thymectomized animals grafted with lymph node fragments demonstrated no significant increase in lymphocyte numbers nor was there a return of immunocompetence as compared to thymectomized controls.     

The migration of lymphocytes across specialized vascular endothelium. IV. Prednisolone acts at several points on the recirculation pathways of lymphocytes

Radioactively labelled thoracic duct lymphocytes from syngeneic rat donors were injected iv into recipients which had been given a continuous iv infusion of prednisolone at 1 mg/hr for 15–18 hr previously. The tissue distribution and recirculation into lymph of the labelled lymphocytes were compared quantitatively in the prednisolone-treated and control recipients by scintillation counting and autoradiography. The most prominent effect of prednisolone was to retard recirculating lymphocytes within the tissues to which they are normally distributed by the blood, namely the bone marrow, the spleen, and the lymph nodes. Although lymphocyte traffic was almost completely frozen by prednisolone, recirculating lymphocytes were not killed. A second effect of prednisolone was to impair the influx of lymphocytes from the blood into lymph nodes. Different groups of lymph nodes varied in the extent to which prednisolone inhibited the entry of lymphocytes, and previous antigenic stimulation completely exempted lymph nodes from this inhibition. Lymphocytes took a longer time to cross the walls of high endothelial venules in the lymph nodes of prednisolone-treated rats. A third effect of prednisolone was to increase the rate at which lymphocytes entered the bone marrow from the blood by crossing sinusoidal endothelium.     

Circulating T and B lymphocytes of the mouse. II. Lifespan

The average lifespan of circulating lymphocytes was investigated by determining the percentage of labeling of thoracic duct lymphocytes (TDL⁵) from mice injected with tritiated thymidine (³HT) for various periods. Percentage of labeling of TDL from normal CBA mice, which consist of approximately 85% T cells and 15% B cells, was found to be directly proportional to the time of ³HT administration. This technique thus failed to demonstrate the presence of more than one population of lymphocytes. Less than 50% of TDL were labeled after ³HT injection for 8 weeks.Percentage of labeling of TDL from nude mice (which consist solely of B cells) was likewise found to be directly proportional to the duration of ³HT injection but occurred at a rate three to four times faster than in non-T cell-depleted CBA mice. Further experiments, in which a marker for B cells was used, allowed the rate of ³HT labeling of B cells to be studied in normal CBA mice. These data corroborated the findings in nude mice and indicated that, with regard to lifespan, thoracic duct B cells consisted of a single population with an average lifespan of 5–7 weeks. Similarly it was calculated that the average lifespan of thoracic duct T cells was in the order of 4–6 months.Studies on the rate of formation of TDL during prolonged thoracic duct drainage of normal CBA mice indicated that the percentage of newly formed cells increased rapidly after 24-hr drainage. The total numbers of newly formed cells, however, were found to remain relatively constant throughout the period of drainage investigated (up to 9 days) except for a transient increase during the second and third day. Newly formed small lymphocytes were found to consist of approximately equal proportions of T cells, B cells, and other “mononuclear” cells which lacked surface markers for either T or B cells. The great majority of large lymphocytes, in contrast, were found to be neither T cells nor B cells and probably belonged to the plasma cell line. In nude mice, production of newly formed lymphocytes during prolonged thoracic duct drainage was found to be very low in comparison with normal CBA mice.     

Mitogenic factor from inbred guinea pigs. II. Properties of the factor

Thymectomized adult rats which have been heavily irradiated and reconstituted with syngeneic bone marrow cells rapidly regain the ability to defend themselves against a primary infection with the intracellular bacterial parasite, Listeria monocytogenes. They do so by a cell-mediated immunological mechanism as evidenced by the protective immunity transferred adoptively by thoracic duct lymphocytes or peritoneal exudate cells from donors infected with this organism. But peritoneal exudate cells from thymus-derived donors convey only a fraction of the immunity transmitted by exudate cells from similarly infected intact rats. Since thymectomized irradiated animals can mobilize their cellular defenses more effectively when they are injected with a modest number of thoracic duct lymphocytes, an effect that cannot be duplicated with a massive infusion of bone marrow, it is argued that thymusdependent lymphocytes or T cells have an influential role in the development of cellular resistance to infection.     

Immunoglobulin on activated T cells detected by indirect immunofluorescence

A high proportion of H2 antigen-activated thymus-derived thoracic duct lymphocytes stained positively with rabbit anti-mouse immunoglobulin reagents as detected by indirect immunofluorescence. In view of the specificity of the reagents used and the fact that T cells from other sources e.g., thymus, were not stained by this technique, it was concluded that the material detected on H-2 antigen-activated thymus-derived thoracic duct lymphocytes was indeed immunoglobulin. When H-2 antigen-activated thymus-derived thoracic duct lymphocytes were cultured in vitro at 37 °C for 18 hr, with or without prior treatment with chymotrypsin, surface immunoglobulin could no longer be detected on the cells. This suggested that immunoglobulin had not been synthesized by the cells but absorbed from elsewhere.     

Lymphopoiesis in the rat. I. The effect of pool size on lymphocyte production

A method is described for measuring lymphopoiesis that enables the production of recirculating and non-recirculating small lymphocytes to be estimated simultaneously. Using this technique, experiments were undertaken to determine whether the production of recirculating cells is influenced by the number present in the recirculating lymphocyte pool. The results suggest that neither a massive lymphocyte transfusion nor depletion of the pool by whole body irradiation or chronic lymph drainage affect the rate at which recirculating small lymphocytes are generated.     

Thymus-derived Cells in Mouse Thoracic Duct Lymph

Thymus lymphocytes injected into neo-natally thymectomized mice were identified in the thoracic duct lymph by their θ antigens and shown to form part of the recirculating lymphocyte pool.     

Circulating T and B lymphocytes of the mouse. I. Migratory properties

Studies on the identity of thoracic duct lymphocytes (TDL⁴) from normal and T cell-depleted mice indicated that as many circulating B lymphocytes were produced by healthy T cell-depleted mice as normal mice. Proportions of T and B cells from the thoracic duct of CBA mice changed markedly during the first 4 days of drainage from 82% T cells and 16% B cells at 12 hr to approximately equal proportions of both classes after 3 days. In absolute terms, T cells were mobilized rapidly by thoracic duct drainage and B cells very slowly. Histologically, this was reflected in a rapid depletion of the T cell-dependent areas of the lymphoid organs. The B cell-dependent areas, in contrast, became depleted of lymphocytes only after drainage for a week or more.The homing properties of circulating lymphocytes were investigated using TDL from normal and T cell-depleted mice as relatively pure sources of T and B cells, respectively. Four hours after injection of ⁵¹Cr-labeled T and B cells, a large proportion of both cell classes were found in the spleen. By 24 hr, many T cells had left the spleen and appeared in the lymph nodes. Such redistribution by B cells, however, was minimal.Intravenously injected T and B cells, labeled with tritiated uridine (³HU), localized specifically in the T and B cell-dependent areas, respectively, of the lymphoid tissues.³HU-labeled T cells were found to recirculate rapidly from blood to lymph. Labeled B cells, in contrast, recirculated only very slowly.     

3H-uridine incorporation as a T lymphocyte indicator in the rat

Although about 70% of rat thoracic duct small lymphocytes labeled readily in vitro with ³H-uridine, only 3–38% of peritoneal exudate lymphocytes labeled. Since exudate cells are mostly B lymphocytes, ³H-uridine in concentrations used were presumed to label the T lymphocyte. Percentages of small lymphocytes that labeled in cell suspensions from various tissues were consistent with other estimates of T cells in those sources: 74.7% in thoracic duct, 70.2% in blood and 65.6% in spleen. When lymphopenia was induced by polyethylene ³²P strips applied to the spleen, a procedure that depletes mostly small recirculating lymphocytes, both labeled (T) and nonlabeled (B) cells were depleted in similar time sequence. Both cell types recovered at a similar rate after the spleen strips were removed. Induction of peritoneal inflammation by PPD in tubercle-bacilli immune rats caused an enhanced lymphocytic exudation but no increase in percentage of labeled (T) lymphocytes.The defect in ³H-uridine incorporation that characterizes the rat B lymphocyte seemed to be relatively specific for that RNA precurser; ³H-cytidine labeled the majority of lymphocytes in peritoneal exudate.     

On the mode of action of thymosin

Thymosin was administered to CBA mice which had been depleted of recirculating small lymphocytes by combining ALS and thymectomy or through lethal irradiation of thymectomised mice reconstituted with syngeneic bone marrow. The population of recirculating small lymphocytes was monitored by determining the numbers of “lymph node localising” cells in the lymphoid organs of treated animals. In no case was there any evidence that thymosin treatment accelerated the recovery of recirculating lymphocytes. Moreover, it was not possible to show that bone marrow cells incubated with thymosin acquired theta-positivity.We conclude that thymosin does not act by augmenting the production of mature recirculating small lymphocytes.     

Generation of macrophage chemotactic activity in situ in Listeria-immune rats.

Macrophage chemotactic activity (MCA) is generated in situ in peritoneal inflammatory exudates induced by antigens of the intracellular parasite, Listeria monocytogenes. Chemotactic and chemokinetic activity is formed locally in response to an immunologically specific signal. In rats that have been immunized adoptively with thoracic duct lymphocytes (TDL) from specifically immunized donors, the production of MCA depends upon stimulation by LMA of exudate-seeking S-phase lymphocytes of their progeny. The sequential appearance, increase, and subsequent decline of MCA in the peritoneal cavity parallels the influx of lymphocytes and precedes maximal recruitment of labeled monocytes from the blood. The MCA response in peritoneal exudates induced in adoptively immunized rats correlates with the level of macrophage accumulation in the peritoneal cavity and at sites of LMA injection in the pinna of the ear. The results suggest that MCA is released locally by antigen-activated lymphocytes and imply that the factor(s) concerned has a purposeful role in the host's defense by promoting the rapid deployment and/or retention of monocyte-derived macrophages in centers of infection.     

Studies on the recirculating cells in the mouse thymus

The cells in the mouse thymus which respond to PHA and have the distribution characteristics of recirculating lymphocytes upon infusion into isologous recipients are markedly enriched 2 days after treatment of mice with proper doses of cortisone acetate. However, a similar increase is not observed in lymph nodes and spleen. During regeneration of the steroidinvoluted thymus, the proportion of recirculating thymic cells disappeared more rapidly than the relative PHA-responsiveness of the cell population. These findings indicate that all PHA-responsive thymocytes may not have recirculating capacity. The recirculating cells present in the thymus and lymph nodes have no tendency to distribute to the thymus, but tend to accumulate in lymph nodes. This fact, together with the finding that in vitro trypsin treatment of the cells temporarily abolishes their lymph node-seeking capacity, are taken as evidence that the recirculating cells in the thymus have undergone their maturation within this organ whereby they have gained membrane receptors which determine their distribution characteristics.     

Nippostrongylus brasiliensis: intestinal goblet-cell response in adoptively immunized rats.

Goblet-cell differentiation was studied in the intestinal epithelium of rats infected with the nematode Nippostrongylus brasiliensis. An increase in the proportion of goblet cells occurred at the time of worm expulsion in rats infected with 1000 or 4000 third stage larvae. Adoptive immunization of infected rats with immune-thoracic duct lymphocytes (TDL) induced extensive goblet-cell differentiation whereas the transfer of immune-TDL into normal rats had no effect. The extent of goblet-cell differentiation in adoptively immunized infected rats was proportional to the number of cells transferred. A goblet-cell response also occurred in adoptively immunized rats harboring implanted “normal” and “damaged” worms but recipients of normal worms which were not given cells were unable either to expel their worm burden or to induce a goblet-cell response. Experiments in which the parasites were expelled with an anthelmintic drug suggested that the goblet-cell increase was not simply a repair process associated with the expulsion of the parasites. In all situations where immune expulsion of the parasites occurred, there was a concomitant rise in the proportion of goblet cells. These experiments suggest that thoracic duct lymphocytes either directly or indirectly regulate the differentiation of intestinal goblet cells.     

The bronchoalveolar lymphocyte: Studies on the life history and lymphocyte traffic from blood to the lung

Although recent work has shed some light on the identity and function of lymphocytes that reside in the bronchoalveolar air space (lung lymphocytes), little is known about the origin and life history of these cells. To determine the proportion of recently divided lung lymphocytes, DA-strain rats were labeled in vivo for 3 days with tritiated thymidine ([³H]dTR). Autoradiographs of lavaged lung and peritoneal cells indicated that a large fraction (44–77%) of lung lymphocytes was labeled and that these values were comparable to the proportion of labeled lymphocytes in peritoneal exudates (61–74%). To determine if some newly divided lung lymphocytes might come via the blood, additional experiments were performed in which rats were labeled ([³H]dTR) in vivo for 7 days. Lymphocytes were then obtained in labeled rats by thoracic duct drainage and were adoptively transferred (by vein) into syngeneic recipients. The percentage recovery of labeled lymphocytes in lung aspirates of recipient rats was determined from cell counts and autoradiographs. These results demonstrate that blood may be a source of recently divided lymphocytes but they do not indicate the relative contribution blood makes toward these cells in the lung.     

Ontogeny of the immune system inSebastiscus marmoratus: Histogenesis of the lymphoid organs and effects of thymectomy

Synopsis The ontogenetic development of the immune system in a marine teleostSebastiscus marmoratus was studied by histological examination and removal of the thymus. The pronephros and the spleen had been differentiated at the time of birth and contained small numbers of haemopoietic cells. In contrast to most vertebrates, the rudiments of the thymus were first visible 1 week post-birth in the dorsoposterior part of the pharynx, the same location as in the adults. However, small lymphocytes first appeared in the thymus of fish at 3 weeks of age, followed by the pronephros at 4 weeks and the spleen at 6 weeks. Complete or partial suppression of the antibody response to sheep red blood cells (SRBC) occurred in fish that were thymectomized at 1.5 months of age and immunized 2 weeks later, and a marked decrease in lymphocytes was observed in the pronephros and spleen. The thymectomy of adult fish also caused reduced serum antibody titres in fish immunized 1 month after the operation. These results suggest that the thymus plays an essential role in the development of the immune system and its functions continue into adult life.     

On the lifespan of virgin T lymphocytes.

To study the lifespan of virgin T lymphocytes, we removed the thymus from adult female mice and then, at various times afterward, tested their ability to mount an immune response to a newly encountered Ag, the male Ag H-Y. We found that unprimed thymectomized mice were able to generate a primary response to H-Y for some time after thymectomy but lost this ability at approximately 6 mo. In contrast, mice that were primed to H-Y just after thymectomy continued to display immunological memory to H-Y for >1 year. These experiments show that primary immune responses disappear in the absence of a thymus.     

The generation of large pyroninophilic cells in the lymphoid tissues of rats infused with cell-free lymph.

The thoracic duct of Wistar strain rats was cannulated during 5 days for studying the effect of selective lymphocyte depletion on the lymphoid tissue. A technique for the continuous infusion of cell-free lymph, whole lymph of Eagle's medium to the rat with the thoracic duct fistula is described in detail. The prolonged drainage of lymph from rats was followed by lymphopenia, sever atrophy of lymphoid tissues and the depletion of small lymphocytes in the thymus-dependent areas of spleen and lymph nodes. The infusion of cell-free lymph into the drained rat resulted in the recovery of the weight of lymphoid tissues and in the massive proliferation and accumulation of large cells with prominent nucleoli and intensely pyroninophilic cytoplasm in the lymphocyte depleted areas of the peripheral lymphoid tissues and thymic cortex. There was histological evidence that the large pyroninophilic cells developed well in the spleen and tended to localize preferentially around the periarteriolar region through the marginal zone bridging channels to the red pulp. The infusion of Eagle's medium was found ineffective in restoring the weight of the lymphoid tissues and in bringing about the proliferation of lymphoid cells. The rats infused with whole lymph showed almost similar findings biologically and histologically to those of sham-operated rats.