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.     

STUDIES ON LYMPHOCYTES

Tritiated thymidine was administered to calves continually for 2 to 8 days via the thymic artery in an attempt to label intensively thymic lymphocytes. Heavily labeled cells which had migrated from the thymus were observed in the spleen, lymph nodes and Peyer's patches. Cell maps were made for the various lymphoid tissues and in all cases the majority of labeled thymic cells were found in the ‘thymus dependent areas’of the spleen and lymph nodes. The number of labeled thymic cells per thousand lymphocytes was highest in the ‘thymus dependent areas’. A few labeled thymic cells were seen in or near the post capillary venules. The labeling pattern in the Peyer's patches was different from that in the spleen and lymph nodes. Labeled thymic cells were not observed in the bone marrow. Heavily labeled cells were not detected in any of the lymphoid tissues of those calves which received continuous intravenous infusion of comparable amounts of tritiated thymidine.     

Ganglioside composition of normal and leukemic bovine lymphocytes

The ganglioside composition of bovine peripheral lymphocytes was shown to change sharply under lymphoid leukemia. In normal lymph, lymph nodes, spleen and blood lymphocytes the major ganglioside is N-glycolylhematoside, whereas in calf thymus lymphocytes appreciable amounts of more polar components (GM1- and GD1a-like gangliosides) were found. In leukemic lymphocytes isolated from the same tissues the hematoside content is decreased, while the amount of more polar gangliosides is increased. Possible causes of the altered ganglioside pattern in leukemic lymphocytes are discussed.     

Terminal galactosyl residues of cell-surface glycoconjugates exposed on both human and murine immature T- and B-cells

Summary Exposure of terminal galactosyl residues on cell-surface molecules as detected by their ability to bind peanut lectin (PNL) is found to be characteristic for immature cortical human and murine thymic lymphocytes. While in prenatal mice PNL staining is found to be uniformly distributed among all thymic lymphocytes, in adult thymi a cortico-medullary gradient is detectable concerning the PNL-binding capacity of thymic cortical lymphocytes, a phenomenon that appears to be correlated to their maturational degree.In secondary lymphatic tissue, i.e. lymph nodes and spleen of man, mouse and rat, strongly labeled cells are found exclusively in germinal centers. Ultrastructurally, these cells could be identified as centrocytes and centroblasts. These observations suggest that exposed galactosyl moieties of cell-surface glycoconjugates are expressed by undifferentiated lymphocytes of both T-and B-cell lineage.Furthermore, it could be shown that PNL-binding properties of immature cells are not restricted to lymphatic tissue but can also be demonstrated on various embryonic cells of non-lymphatic origin in distinct developmental stages. Thus, they might have a fundamental significance in the course of maturation processes.Supported by the Deutsche Forschungsgemeinschaft     

Purification and tissue distribution of human thymidine phosphorylase; high expression in lymphocytes, reticulocytes and tumors

Thymidine phosphorylase (dThdPase) is an enzyme involved in pyrimidine nucleoside metabolism, but little is known about its physiological functions. We purified dThdPase from human placenta and used it for antibody preparation. The purified material appears as a single band at 55,000 dalton on sodium dodecylsulfate-polyacrylamide gel electrophoresis. We obtained a specific antibody raised in rabbits that detected a single polypeptide with a molecular weight of 55,000 dalton in the post nuclear homogenates of several human tissues, on immunoblotting. Using the same technique, dThdPase was highly expressed in the liver, lung, spleen, lymph nodes and peripheral lymphocytes. Immunohistochemical staining revealed that macrophage-like cells contained a much higher amount of dThdPase than parenchymal cells in the liver and lung. dThdPase was found to be highly expressed in T- and B-cell-type malignant lymphoma cells, but low in lymphoblastic and myeloblastic leukemia cells. We also found that carcinomas in the stomach, colon and ovary contained higher amounts of this enzyme than non-neoplastic regions of the tissues. These data suggest that dThdPase plays a role in proliferation and/or differentiation of leukocytes and in cancer proliferation.     

Immunomorphological localization of adenosine deaminase in rat tissues during ontogeny

Summary Immunomorphological methods were used to localize adenosine deaminase in tissues of the rat at different stages of ontogeny. In the thymus, lymphocytes began to express significant amounts of the enzyme with the appearance of demarcation between the cortex and medulla at 17 days of gestation. At any stage of ontogeny studied, strong adenosine deaminase staining was seen predominantly in cortical thymocytes. In the spleen and lymph node, the enzyme was initially detected in T cell areas, whereas primary follicles did not show positive adenosine deaminase staining. During further development, the enzyme was demonstrated in some lymphocytes of germinal centres and plasma cells. In the duodenum, epithelial cells of villi and the neck of crypts showed positive adenosine deaminase staining whereas no staining for the enzyme was observed in the epithelial cells of the base of crypts. Strongly positive staining for adenosine deaminase appeared in plasma cells of the lamina propria by four weeks after birth. The transient positive reaction for the deaminase could be recognized in epithelial cells of tubules of the kidney during late foetal and early postnatal development. The tubules of adult rats did not stain for the enzyme. In the cartilage of 15-day foetuses, positive adenosine deaminase staining was seen only in perichondrial cells and hypertrophic cells. Kuppfer cells in the liver and endothelial cells of blood vessels stained positively for the enzyme at every stage of ontogeny studied.     

Purine metabolic enzymes in lymphocytes. I. Adenosine deaminase and purine nucleoside phosphorylase activities in mouse lymphocyte subpopulations

The distribution of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities in lymphoid organs and lymphocyte subpopulations in mice, and the effect of phytohemagglutinin P (PHA-P) and concanavalin A (Con A) on the enzyme activities were studied. ADA activity was distributed equally in cells from all organs used and no mouse strain differences were observed. In contrast, PNP activity varied with the mouse strain, being highest in C57BL/6 mice and lowest in BALB/c mice, and with the organ in ICR mice, being high in peripheral blood lymphocytes and spleen lymphocytes, low in mesenteric lymph node cells and absent or very weak in thymus cells. T and B lymphocytes were prepared from spleen of ICR mice. High ADA activity was found in both T and B lymphocytes, whereas PNP activity in the T lymphocytes was about one-third of that in the B lymphocytes. PNP activity in thymus cells was increased to the normal level of T lymphocytes in the spleens by cultivation without stimulant. The development of PNP activity in thymus cells was partially inhibited by Con A but was not affected by PHA-P. ADA activity in thymus cells was enhanced by in vitro stimulation with PHA-P but not with Con A. In contrast, in spleen lymphocytes the development of ADA activity was enhanced by stimulation with PHA-P and Con A, and that of PNP activity was enhanced by PHA-P but not by Con A.     

Interaction of the lymph node and splenic lymphocytes of mice in inactivation of allogeneic hematopoietic stem cells

A study was made of interaction of mouse spleen and lymph node lymphocytes in inactivation of allogeneic stem cells. It was established that T lymphocytes of the lymph nodes and spleen lymphocytes do not interact on combined administration; their action is of additive nature. B lymphocytes of the lymph nodes have a regulating activity both in respect to T lymphocytes of the lymph nodes and lymphocytes of the spleen. The stem cells serve as target. Depending on the stem cells/B lymphocytes ratio B lymphocytes are capable of exerting either helper or suppressor action.     

The influence of strong transplantation antigens (Ag-B) on lymphocyte migration in vivo.

The tissue localization of syngeneic thoracic duct lymphocytes was compared to that of allogeneic cells in four rat strain combinations differing at the Ag-B locus (HO → DA, DA → HO, AO → HO, HO → AO). Dual isotope labeling with [³H]uridine and [¹⁴C]uridine was applied in order so that the distribution of allogeneic and syngeneic cells could be followed in one recipient. During the first couple of hours after iv injection, allogeneic lymphocytes usually migrated as easily into the various tissues as did syngeneic cells. However, after 24 and 48 hr, a reduced amount of label associated with allogeneic cells was often measured in the tissues. This reduction differed in magnitude in the different strain combinations and was most pronounced in the lymph nodes. A reduced number of allogeneic cells also appeared in the thoracic duct. By contrast, no reduced localization of allogeneic lymphocytes was measured in the draining popliteal lymph nodes late after sc injection. In preimmunized animals allogeneic cells were rapidly removed from the blood and therefore failed to localize in the lymphoid tissues. Furthermore, the lymph node localization of allogeneic cells was more like that of syngeneic cells in splenectomized rats, as well as in irradiated recipients (when the irradiation was given shortly before cell transfer). It is concluded that transplantation antigens play no essential role in the interaction between recirculating lymphocytes and the venous endothelium at the sites where the large-scale physiological emigration of the cells takes place (the HEVS of the lymph nodes and the marginal zone vessels of the spleen). The elimination of allogeneic cells is found later; it probably takes place in the lymph nodes and spleen. Possible mechanisms responsible for this rapid removal of allogeneic lymphocytes in nonimmunized recipients are discussed.     

STUDIES ON LYMPHOCYTES

Continuous extracorporeal irradiation of the circulating blood (ECIB) of from 3 to 501/2 hr duration was used to study in the calf the differential depletion of lymphocytes from spleen, lymph nodes and thymus as compared to blood and thoracic duct lymph. The cell content of tissues was measured by planimetry and/or test point analysis. Lymphocyte depletion by ECIB from various lymphoreticular organs, and from different areas within a given organ, was less than in the circulating blood or the thoracic duct lymph and varied from one site of a lymphoreticular organ to another. The degree of depletion with time followed an exponential function with at least two components. The first component corresponded to a relatively rapid fall and the second to a very slow reduction in lymphocyte content. The former is related to the elimination of an easily mobilizable pool of lymphocytes while the latter corresponds to a more sessile mass of lymphocytes which exchange with blood lymphocytes very slowly. Elimination of the easily mobilizable pool of lymphocytes by ECIB from all tissues studied was observed within 10–15 hr, indicating that the rate of exchange with blood is similar for this group of cells in various lymphoreticular tissues. The size, however, of the easily mobilizable vs the more sessile pools of lymphocytes may vary considerably, the best estimates for the former being as follows (in per cent of total lymphocyte mass): lymph node medulla, less than 10%; lymph node cortex plus paracortical zone, 18% (depletion mainly paracortical); red pulp of the spleen, 37%; densely populated white pulp of the spleen, 55%; and loosely populated white pulp of the spleen, 60%. In comparison, the approximate fractions of lymphocytes originating fromthe easily mobilizable pools in various lymphoreticular tissues plus the cells already circulating a t the onset of EClB correspond to 64% for the thoracic duct lymph and 78% for the circulating blood respectively. These findings are discussed in relation to production, recirculation and life span of lymphocytes, and immune reactions.     

Expression of the rat CD26 antigen (dipeptidyl peptidase IV) on subpopulations of rat lymphocytes.

The T cell activation antigen CD26 has been recently identified as the cell surface ectopeptidase dipeptidyl peptidase IV (DPP-IV). DPP-IV is found on many cell types, including lymphocytes, epithelial cells, and certain endothelial cells. The MRC OX61 monoclonal antibody (MAb) which specifically recognises rat DPP-IV was used to examine the expression of CD26/DPP-IV on rat lymphocytes. The molecular nature of the antigen was examined by immunoprecipitation from thymocytes, splenocytes, and hepatocytes. Analysis by one- and two-dimensional gel electrophoresis indicated that the native form of CD26 includes a 220-kDa homodimer. On tissue sections MRC OX61 MAb stained nearly all thymocytes and in the spleen and lymph nodes predominantly stained the T cell areas. However, in immunofluorescence experiments OX61 stained 80 to 87% of lymph node cells and 78 to 85% of spleen cells. Furthermore, two-colour immunofluorescence analysis of the CD4+, CD8+, and Ig+ lymphocyte subsets indicated that only 2 to 5% of each of these subsets lacked OX61 staining. Spleen cells and thymocytes of both CD4+ and CD8+ subsets stained much more intensely with OX61 after these cells were stimulated with phytohemagglutinin. These findings indicate that rat CD26 antigen expression is not confined to the T cell population as has been suggested, but also occurs on B cells, and is increased on T cells following their activation.     

The distribution of adenosine deaminase among lymphocyte populations in the rat.

Adenosine deaminase (ADA) activity was determined in young rat lymphocyte populations. The ADA-specific activity (per 10(8) cells and per milligram protein) was 3- to 10-fold higher in thymocytes than in lymphocytes from thoracic duct, lymph node, spleen, and bone marrow. The high ADA activity in thymocytes appeared to be preferentially associated with cortical thymocytes. Enrichment or depletion of cortical thymocytes by density gradient centrifugation, cortisone treatment, or selective lysis with anti-Thy-1 plus complement resulted in parallel increases or decreases in ADA levles. These results also suggested that medullary thymocytes have ADA levels similar to those of peripheral lymphocytes. "Immature" cortical thymocytes and thymocyte progenitors appeared to have low ADA activity; low enzyme levels were found in fetal thymus at 16 days of embryonic life, in the early phases of thymus regeneration, and in a "null" cell population isolated from bone marrow. This study demonstrates that ADA activity varies markedly during T lymphocyte differentiation and suggests that fundamental differences in nucleotide metabolism may exist in T cells at different stages of development.     

Characterization of the endogenous deoxyribonuclease involved in nuclear DNA degradation during apoptosis (programmed cell death).

Cell death by apoptosis occurs in a wide range of physiological events including repertoire selection of lymphocytes and during immune responses in vivo. A hallmark of apoptosis is the internucleosomal DNA degradation for which a Ca2+,Mg(2+)-dependent endonuclease has been postulated. This nuclease activity was extracted from both rat thymocyte and lymph node cell nuclei. When incubated with nuclei harbouring only limited amounts of endogenous nuclease activity, the ladder pattern of DNA fragments characteristic of apoptosis was induced. This extractable nucleolytic activity was immunoprecipitated with antibodies specific for rat deoxyribonuclease I (DNase I) and was inhibited by actin in complex with gelsolin segment 1, strongly pointing to the presence of a DNase I-type enzyme in the nuclear extracts. COS cells transiently transfected with the cDNA of rat parotid DNase I expressed the enzyme, and their nuclei were able to degrade their DNA into oligosome-sized fragments. PCR analysis of mRNA isolated from thymus, lymph node cells and kidney yielded a product identical in size to that from rat parotid DNase I. Immunohistochemical staining with antibodies to rat DNase I confirmed the presence of DNase I antigen in thymocytes and lymph node cells. The tissue distribution of DNase I is thus extended to tissues with no digestive function and to cells which are known to be susceptible to apoptosis. We propose that during apoptosis, an endonuclease indistinguishable from DNase I gains access to the nucleus due to the breakdown of the ER and the nuclear membrane.     

Cytotoxicity of effector cells of the peripheral blood, lymph nodes and spleen in Hodgkin's disease

The mononuclear cells and T-lymphocytes of the blood, spleen and lymph nodes from 83 patients with Hodgkin's disease and 50 healthy donors were tested in assays for lectin-dependent (LD) and natural killer (NK) cytotoxic activity (CTA). On an average, peripheral blood T cell LD-CTA of patients did not differ from that of the donors. However, the CTA appeared to be dependent on the stage of the disease; in the IVth stage LD-CTA was decreased 2-fold. The LD-CTA was also dependent on the histological type of disease and the lowest level of LD-CTA (50% of the control level) was associated with the "lymphocyte depletion" type. The CTA of T-lymphocytes from the affected areas of the patients' spleen was more marked than that of the unaffected areas. Spleen cell CTA showed no other correlations. The CTA of lymphocytes from the affected lymph nodes was drastically lower than CTA of blood and spleen lymphocytes. The NK activity of the patients' blood and spleen lymphocytes was twice as less as the control level (healthy donors) and did not correlate with a stage and/or a histological type of the disease. It was assumed that in Hodgkin's disease the specific antitumor immunity remains mostly within normal and is decreased only in the last, terminal stage of the disease.     

Detection and quantification of IgM(+) lymphocytes in fetal lamb spleen, liver and lymph nodes by flow cytometry

The first stage in Peyer's patch development in the fetal lamb is characterized by the colonization of the rudimentary Peyer's patches by precursor cells expressing the IgM surface receptor. In the fetal lamb, the spleen has been implicated as the source of gene-rearranged IgM(+) B lymphocytes. This study was intended to quantitate IgM(+) lymphocytes in the spleen, lymph nodes and liver of fetal lambs at various gestational ages between 63 and 110 days using flow cytometry. Flow-cytometric analysis revealed that IgM(+) lymphocytes were rare in the liver being consistently less than 1% at every gestational age examined. IgM(+) lymphocytes were detected in the spleen (mean 9.18%) and prescapular lymph nodes (mean 11.89%) as early as 63 days. In both spleen and lymph nodes, the highest representation of IgM(+) lymphocytes occurred between 70 and 86 days gestation. The highest mean percentage of IgM(+) lymphocytes was observed in the spleen (22.63%) and lymph nodes (17.02%) at 75 days gestation. From 98 days onwards, B-lymphocyte density gradually decreased in both spleen and prescapular lymph nodes. This study indicates that substantial populations of IgM(+) lymphocytes were present in both the spleen and prescapular lymph nodes from 70 days gestation and implies that both of these locations could be potential sources for the normal colonization of the ileal Peyer's patches.     

Analysis of lymphoid and dendritic cells in human lymph node, tonsil and spleen. A study using monoclonal and heterologous antibodies

The distribution of lymphoid and dendritic cells in human reactive lymph nodes, tonsils and spleens was examined by means of an indirect immunoperoxidase technique, using a panel of monoclonal and heterologous antibodies. The antibodies used were directed against antigens present on T cell subsets (Leu1, leu2a, Leu3a, TA1, OKT6), various types of B cells (BA1, BA2, HLA-DR, CR1) and cells of the mononuclear phagocyte system (alpha HM1, TA1, CR1, OKM1, NA 1/34). In the lymph node and tonsil Leu3a-positive cells (T-helper/inducer phenotype) and Leu2a-positive cells (T-suppressor/cytotoxic phenotype) are found in the thymus-dependent or T-cell area; in the spleen Leu3a-positive cells are found mostly in the periarteriolar lymphocyte sheath (PALS), while Leu2a-positive T-suppressor/cytotoxic cells are almost completely restricted to the cords of Billroth in the red pulp. The cells in the mantle zone of germinal centres and in the primary follicles in lymph nodes, tonsils and spleens have B-cell properties (BA1-, HLA-DR-, and CR1-positive). The cells in the germinal centres show a similar staining pattern (HLA-DR-, and partly CR1-positive). Follicles and T-cell-dependent areas have specific dendritic cells, each with a specific staining pattern: the dendritic reticulum cell (DRC) of the follicle stain with CR1, HLA-DR, BA2 and alpha HM1; the interdigitating cell of the T-cell areas in the lymph node, tonsil and spleen stain with HLA-DR and BA1. Moreover, large dendritic OKT6-positive cells are found in the T-cell areas of some of the peripheral lymph nodes, and are probably Langerhans cells. It is concluded that human lymph nodes and tonsils have an identical compartimentalisation, clearly differing from the spleen in cellular organization.     

Tissue and cellular distribution of alpha-L-iduronidase in the pig

We investigated the alpha-L-iduronidase activity of various pig tissues. Furthermore, we examined the tissues using antibody, enzyme immunoassay (EIA), and immunohistochemical methods. The amounts of enzyme measured by the EIA method in the various tissues were proportional to their enzyme activities and also to their immunohistochemical characteristics. The tissues could thus be classified into three groups: a high enzyme activity group composed of the liver, kidney, and spleen; a moderate activity group comprising the lung, lymph nodes, stomach, ileum, colon, and pancreas; and a low activity group consisting of the heart, diaphragm, iliopsoas muscle, cerebrum, cerebellum, and skin. The molecular weight of the enzyme in each tissue did not reveal any heterogeneity, having two components of 70 KD and 62 KD by Western blot analysis. Immunohistochemically, alpha-L-iduronidase was strongly detected in the lysosomal membranes of cells of the mononuclear phagocyte system, epithelial cells of the proximal tubules in the kidney, and some blastic cells, whereas hepatocytes revealed weak positive reactions. The tissue and cellular distribution of the enzyme appeared to have a close relation to tissues that manifest or are affected by alpha-L-iduronidase deficiency.     

Persistent infection of rabbits with bovine immunodeficiency-like virus.

Chronic infection of rabbits was induced by a single intraperitoneal injection of bovine immunodeficiency-like virus (BIV)-infected cells. Ten BIV-infected animals were monitored serologically for up to 2 years. Results of serologic and virus rescue assays indicated that all animals became infected and demonstrated a rapid and sustained BIV-specific humoral response. BIV was rescued by cocultivation from spleen, lymph nodes, and peripheral blood leukocytes of infected animals. Viral DNA in immune tissues was confirmed by polymerase chain reaction amplification of BIV sequences. These data and specific immunohistochemical staining of mononuclear cells of the spleen for BIV antigen suggest that the infection is targeted to immune system cells.