Thoracic Duct Cells Research Articles

Are Actinomyces Viscosus Antigens B Cell Mitogens?

Abstract An extracellular heteroglycan (ECHG) and a sonicated cell supernatant (SCS) of Actinomyces viscosus Ny 1 induced strong lymphocyte proliferation. This was shown with spleen and thoracic duct cells from germfree rats and confirmed with cells from conventional “nude” mouse spleens. Spleen cells developed direct plaque-forming cells against densely coupled TNP-SRBC. The mitogenic property of ECHG was diminished considerably after mild alkaline hydrolysis for lymphocytes from rat spleens and was totally abolished for cells from “nude” mouse spleens. These results suggest that ECHG and SCS have B cell mitogenicity.

Maturation of B Lymphocytes in Rats

Abstract We examined the ability of large and small thoracic duct cells obtained from Lewis rats primed to DNP to restore the adoptive secondary anti-DNP response in irradiated syngeneic hosts given an excess of T helper cells. The large cells were four to five times more active on a per cell basis than were the small cells. However, the large cells constitute only 7 to 8% of the thoracic duct cells and, therefore, make a minor contribution to the restorative activity of the unfractionated cells. Pretreatment of thoracic duct cell donors with high specific activity 3H-TdR for 48 hr before cannulation markedly reduced the memory B cell activity of the large cells, but had little effect on that of small cells. In addition, the activity of the large cells diminished with time after primary immunization, but that of the small cells remained stable. Immunofluorescent staining of the large and small cells for surface IgM and IgG, and subsequent sorting on the fluorescence-activated cell sorter (FA CS), showed that surface IgM was present on large memory B cells, and that IgG was present on small memory B cells. The experimental results suggest that two subpopulations of memory B cells in the thoracic duct lymph differ by size, rate of turnover, persistence after primary immunization, and class of surface IgG.

Mixed Leukocyte Reaction in Rats. Effect of Immunization with Bovine Encephalitogenic Protein and Freund's Complete Adjuvant

Rats of certain strains immunized with bovine encephalitogenic protein (EP) in Freund's complete adjuvant develop an impairment of the mixed leukocyte reaction (MLR) similar to that seen in rabbits with experimental autoimmune encephalomyelitis and in humans with certain diseases, including multiple sclerosis. The rats mount a cell-bound response to EP, but encephalomyelitis does not develop. The component causing the impairment was analysed in a culture system using inbred rat strains and F1 hybrids, thoracic duct cells as a source of lymphocytes, and blood as a supplement to the cultures. In normal rats, it was shown that the effects of responding and of stimulating lymphocytes could be separated and that the supportive action of the added blood was probably due to macrophages (monocytes); also that the added blood could in many experiments be replaced with 2-mercaptoethanol (2-ME). The impairment present in immunized rats is at least largely due to a defective supportive activity of the blood (monocytes) and can be restored with 2-ME. The results argue that the MLR impairment seen in immunized rats is due to a faulty macrophage function.

Differences between virgin and memory IgM-antibody-forming-cell precursor B-cells, and correlations with the heterogeneity present in B-cell populations from unimmunized mice

Previous in vitro studies showed that a large proportion of DNP-reactive B-cells in the spleens of unimmunized mice, unlike B-cells reactive to fowl gamma globulin (FGG), did not adhere to glass-bead columns. B-cell reactivity was assessed by challenge with dinitrophenylated-polymerized flagellin (DNP-POL) or FGG in the presence of POL, both responses being thymus-independent. Now we have shown that when donor mice are immunized with FGG, a greater proportion of FGG-reactive B-cells becomes non-adherent and the column filtrates give an IgM anti-FGG response. This indicates then that the adherence properties of the IgM-producing antibody-forming-cell (AFC)-precursor differed in virgin and immunized B-cell populations. In vitro testing of the thoracic duct cell (TDC) population of normal and immunized mice revealed parallels between thoracic duct B-cells and the non-adherent splenic B-cell population. Thus while the in vitro anti-DNP response of thoracic duct lymphocytes from normal mice reached levels little below those of spleen cells, the anti-FGG response was much lower. However when TDC were taken from mice immunized with FGG, the anti-FGG responses were much higher. Thus a parallel was demonstrated both in terms of non-adherence to glass and presence in the thoracic duct, of a portion of the DNP-reactive B-cell population in unimmunized mice and the FGG-reactive population in FGG-immunized mice. This suggested that the non-adherent DNP-reactive B-cells in unimmunized mice may represent B-cells with experience of cross-reacting antigens. The significance of this heterogeneity of the B-cell populations reactive to certain antigens in unimmunized mice is discussed with particular reference to recent apparently contradictory findings in regard to B-cell activation.

The Elaboration of Leukotactic Mediators during the Interaction between Parental-Type Lymphocytes and F1 Hybrid Cells

Mixed leukocyte cultures consisting of white blood cells from (Lewis times BN) F1 hybrids and Lewis parents produced monocyte chemotactic factor. Elaboration of this material preceded incorporation of 3H-thymidine. Local graft vs host (GVH) reactions were induced by subcapsular injection of parental thoracic duct cells into F1 hybrids. Homogenates from these kidneys, but not from kidneys injected with syngeneic thoracic duct cells, contained monocyte chemotactic factor. Little or no neutrophil chemotactic factor was present. Ultracentrifugal analysis of the monocyte chemotactic factor indicated a distribution similar to that found previously in culture fluids of lymphoid cells stimulated by soluble antigens. Differential counts of inflammatory cells extracted in suspension form from kidneys undergoing a GVH reaction indicated the majority of cells to be lymphocytic in type, but with a significant proportion of monocytes. Virtually no neutrophils were present. These findings indicate that a monocyte chemotactic factor is produced by cultures of parental leukocytes stimulated by semiallogeneic cells and that a similar factor appears in GVH reactions in the rat kidney. This chemotactic factor may be relevant to the character of the cellular exudates.

Antigenic relationship between medullary thymocytes and a subpopulation of peripheral T cells in the rat: Description of a masked antigen

Using differentially absorbed rabbit antisera to rat thoracic duct cells, an antigen is described which normally is expressed on the surface of T cells in thoracic duct lymph and lymph node, but which exists in a masked form on medullary thymocytes and apparently not at all on cortical thymocytes. This antigen is termed the rat masked thymocyte antigen (RMTA). RMTA on medullary thymocytes can be unmasked mechanically by sectioning in a cryostat or enzymatically by treating with neuraminidase. Trypsin destroys or removes RMTA. Nearly all the T cells in thoracic duct lymph and lymph node are RMTA +, whereas only 58–66% of T cells in spleen are RMTA +. RMTA + T cells, which are cortisone resistant, reside in the paracortex and periarteriolar sheath regions of lymph node and spleen. RMTA − T cells, which are cortisone sensitive, appear to reside in the red pulp of spleen. The results suggest that (i) two antigenically distinct populations of T cells exist in the rat, RMTA + and RMTA − T cells, (ii) medullary thymocytes are the immediate precursors of RMTA + T cells, and (iii) cortical thymocytes may be the immediate precursors of RMTA − cells.

Maturation of B Lymphocytes in the Rat

Abstract Thoracic duct and spleen cells of normal (unimmunized) adult rats were fractionated according to size by 1 × G velocity sedimentation. Fractions were tested for their ability to restore the adoptive antibody response of irradiated hosts to horse spleen ferritin. A constant source of T cells (small numbers of unfractionated thoracic duct cells) was added to each fraction in order to monitor the B cell activity of the latter. Although large and small cell fractions of the spleen showed restorative activity, only the small cell fractions of the thoracic duct lymph showed activity. The turnover rate of the spleen cell fractions was determined by treating donors with high specific-activity 3H-thymidine for 48 hr before splenectomy. Rapidly dividing cells are preferentially killed by this treatment. The results suggest that a considerable proportion of large, intermediate, and small virgin B cells turn over within 48 hr. The cell surface of the various spleen cell fractions was examined for the presence of immunoglobulin (Ig) and a receptor for complement. The percentage of Ig-bearing cells in the large cell fractions was similar to the percentage of cells bearing IgM and a receptor for complement. However, the majority of Ig-bearing cells in the small cell fractions did not show the latter two surface markers. Experiments with the fluorescence-activated cell sorter showed that the large functionally active B cells bore surface IgM. The experimental findings suggest that there are subpopulations of virgin B cells in the spleen of the adult rat which differ with respect to size, migration pattern, turnover rate, and cell surface characteristics. The relationship of these cells to one another is discussed in the framework of an antigen-independent model of B cell maturation in the rat.

A sensitive microelectrophoretic method for measuring the anti-platelet activity of horse anti-human lymphocyte serum

The anti-platelet activity of horse sera raised against human peripheral or thoracic duct lymphocytes was measured by three methods, platelet agglutination, clot retraction and the lowering of the electrophoretic mobility of platelets. The last method proved the most sensitive by a factor of at least 10. No activity was detected in antisera raised against thoracic duct cells, whereas all antisera against peripheral cells had significant titres. The source of the antigen responsible is discussed.

Evaluation of Oxisuran as an Immunosuppressive Agent

SummaryOxisuran, 2-[(methylsulfinyl)-acetyl]pyridine, is a new immunosuppressive agent which is reported to act as a differential inhibitor of cell-mediated immunity. Published studies have shown that it prolongs skin graft survival in mice, rats, and dogs. The present experiments demonstrate that it is also an immunosuppressive agent in the transplantation of other organs. Oxisuran produced significant prolongation of functional survival time of heterotopic cardiac allografts in rats. In addition, using a rat kidney-transplant model, there was significant reduction of in vitro cytotoxicity of thoracic duct cells against target kidney cells, whereas the humoral antibody response was unchanged.We thank Dr. H. H. Freedman of Warner-Lambert Research Institute, Morris Plains, NJ, for helpful advice and data concerning oxisuran and for generously supplying us with this immunosuppressive agent.

The effect of chronic thoracic duct drainage on cell antigenicity.

AbstractThe surface antigenic topography of thoracic duct cells is described. The cellular population is a heterogeneous one and accounts for the great variety of immunological functions in which these cells participate. Since the lymphatic system is an important route for tumor dissemination, the identification of circulating cells in the lymph by their antigenic confirmation may prove to be a vehicle for the early detection and treatment of cancer.

Maturation of B lymphocytes in the rat. I. Migration pattern, tissue distribution, and turnover rate of unprimed and primed B lymphocytes involved in the adoptive antidinitrophenyl response.

The migration pattern, tissue distribution, and turnover rate of unprimed and primed B lymphocytes involved in the adoptive anti-DNP response was studied. The adoptive primary response restored by unprimed spleen or thoracic duct cells passaged through an intermediate host (intravenous injection and subsequent collection in the thoracic duct lymph) was markedly diminished as compared with that restored by unpassaged cells. On the other hand, the adoptive response restored by passaged spleen or thoracic duct cells from DNP-primed donors was greater than or the same as that restored with unpassaged cells, respectively. This suggests that unprimed B cells change from nonrecirculating to recirculating lymphocytes after exposure to antigen. Studies of the adoptive anti-DNP response restored by unprimed or primed bone marrow cells showed little change in the time-course or amplitude of the response restored by either population of cells. The relative inability of marrow cells to carry immunological memory was related to the inability of recirculating memory cells to penetrate the marrow. The turnover rate of unprimed and primed B cells was investigated by treating the cell donors with [(3)H]thymidine for 48 h before removal of thoracic duct or spleen cells. The adoptive anti-DNP response restored by unprimed or primed cells was not affected by [(3)H]thymidine treatment. This indicates that both populations of cells turn over slowly. However, our previous studies show that unprimed B cells involved in the adoptive antibody response to ferritin turn over rapidly. The different findings are discussed in the context of antigen-dependent B-cell maturation.

Thymus-dependent cytotoxic lymphocytes in the rat.

AbstractThe nature of effector cells mediating cytotoxicity in vitro in a rat tumor allograft model using 51Cr release assays has been investigated. On the basis of a series of in vitro cell depletion techniques including glass absorption, removal of Fc receptor‐bearing cells and lysis of thymus‐dependent lymphocytes with a cytotoxic rabbit anti‐thymus cell sera, the predominant effector cell in this situation appears to be a thymus‐dependent lymphocyte. The main difference from previous published studies in mice appears to be the more rapid evolution of cytotoxic activity in the rat and the difficulty in detecting marked cytotoxic activity in the thoracic duct cells. Determination of the antigenic specificity of these cytotoxic cells by an inhibition assay suggests that their specificity is directed solely to transplantation antigens on the tumor cell.

Graft-vs.-host reaction in tissue culture.

The primary purpose of this study has been to validate the in vitro graft-vs.-host reaction as an experimental system. Time-dose studies have been presented for cells obtained from spleen, thymus, cortisone-treated thymus, inguinal lymph node, mesenteric lymph node, thoracic duct, and bone marrow cells. Both the degree of splenomegaly and the onset of spleen enlargement were found to be dependent on the number and source of cells tested. The effect of several immunosuppressive agents was examined. Amantadine was found to suppress completely the graft-vs.-host reaction in vitro when present at a concentration of 75 microg/ml. Pretreatment of effector cells with mitomycin C prevented their subsequent ability to cause a graft-vs.-host reaction. The effect of X irradiation on immunocompetence of spleen cells in vitro paralleled the known effect of irradiation on in vivo immunocompetence. Preimmunization did not increase the number or effectiveness of immunocompetent cells when measured under standard in vitro conditions. Preimmunization did, however, permit persistence of immunocompetence after immunosuppressive doses of X irradiation. Studies using congenic lines, moreover, indicated that the preimmunization effect could be demonstrated in strain combinations differing only in factors determined by the H-2 complex of genes. A weak graft-vs.-host reaction could be detected in strain combinations not involving differences at the H-2 locus. The potential of the in vitro graft-vs.-host reaction as a highly reproducible, quantifiable, internally controlled, and experimentally accessible system for study of such critical problems as cell differentiation and cell interactions is discussed.

Studies of Thoracic Duct Lymphocytes of Mice

Abstract Thoracic duct cells have a surface antigenic configuration determined by cytotoxic testing that is positive for H-2d, θ, Ly-A, MSLA, κ, and µ antigens and negative for TL and PC antigens. Comparison of these results with other lymphoid elements, benign and malignant, reveals that thoracic duct cells resemble lymph node lymphocytes rather than thymocytes. Quantitative absorption analysis showed similar concentrations of the H-2d, θ, and Ly-A antigens of thoracic duct and lymph node lymphocytes. The findings suggest that the thoracic duct population is a composite of both thymic and bone marrow-derived cells. This antigenic heterogeneity is supported by both functional and morphologic studies.

Ontogeny of cellular immunity: Size and turnover of rat thymocytes responsive to in vitro stimulation

Velocity sedimentation of thymus cell suspensions prepared from Wistar rats of different ages has been used to separate cells mainly on the basis of size. Large cells, many in division, sediment faster than the major population of small thymocytes and can be separated from them on this basis. In vitro responsiveness to mitogens has been found to be associated with the minor populations of larger cells, particularly the medium-large cells (230–270 μm 3). The mitogen-responsive populations also contain cells which show a high autonomous DNA synthesis. The size distribution of thymus cells in neonatal animals is more varied than in adults and there is frequently a higher proportion of large cells. The mitogen-responsive cells were again found among the fast sedimenting large cells and the highest responses were seen with the very largest cells (approximately 450 μm 3). The cells in both adult and neonatal animals which have the capacity to respond to mitogens in vitro are probably larger in size than the normal peripheral recirculating thoracic duct cells. The major population of normal small thymocytes showed a much lower spontaneous uptake of 3H-thymidine and did not respond to mitogens.

EFFECT OF IRRADIATION ON ESTABLISHED DELAYED HYPERSENSITIVITY

When rats immunized with bovine serum albumin in complete adjuvant were irradiated 10 days later with 800 rads their ability to display a positive skin reaction to the test antigen was suppressed for 4–5 days. Thereafter recovery was evident in spite of persisting low blood leucocyte levels. Specific cells were demonstrated in the animals by migration inhibition of peritoneal cells two days after irradiation, when skin reactions were negative. On the other hand transfer of normal bone marrow cells corrected the ability to mount skin reactions but spleen, thoracic duct or lymph node cells did not. It seems that post‐irradiation suppression of skin reactions does not depend on damage of specific cells but depends on shortage of a bone marrow derived cell type participating in skin reactions as a nonspecific component.

Rabbit anti rat lymphocyte serum: in vitro antimacrophage activity of different types of antisera and relationship to immunosuppression.

Antimacrophage activity has been demonstrated in antilymphocyte sera raised with thoracic duct, thymus, and spleen cells. Comparison of various anti spleen cell antisera raised with different antigen doses (1 × 106 ‐ 1 × 108 cells) and immunization schedules (2, 3, 4, 6 and 8 immunizations) was carried out. The presence and degree of antimacrophage activity was related to the cell dose and even more so to the number of immunizations. Antisera with strong antimacrophage activity were less able to prolong the survival of skin allografts than antisera with only moderate antimacrophage activity, the lymphocytotoxic titres being similar. Possible mechanisms for the presence of antimacrophage activity in antilymphocyte sera and in vivo implications of such activity are discussed.

Influence of tumour growth on the evolution of cytotoxic lymphoid cells in rats bearing a spontaneously metastasizing syngeneic fibrosarcoma.

Regional and distant lymph node cells, thoracic duct cells and peripheral blood lymphocytes from rats bearing a spontaneously metastasizing and apparently non-immunogenic sarcoma were assayed for cytotoxic activity on microcultures of tumour cells at 7, 14 and 21 days of tumour growth. In the regional lymph nodes detectable cytotoxicity was present at 7 days and the overall activity remained constant at 14 and 21 days. At Day 7 of tumour growth the cytotoxic cell population in the regional node was tumour specific in its cytotoxic effect, very radiosensitive and could not be removed by nylon wool column purification. In contrast the cells in the regional nodes at Day 21 were nonspecifically cytotoxic and could be completely removed by nylon wool treatment. In the peripheral blood, cytotoxic lymphoid cells not removed by nylon wool, were detectable at all stages of tumour growth. The thoracic duct lymph cells were, however, without cytotoxic activity throughout the period of tumour growth studied. Distant lymph node cells were assayed for cytotoxicity and it was found that they acquired significant cytocidal properties only late in tumour growth. The sera from tumour-bearing rats were tested for inhibitory activity on the cytotoxicity of Day 7 regional lymph nodes from tumour-bearing rats. It was found that a specific inhibitor appeared in the serum and that its activity increased with tumour growth. The possible contributions of the changes in lymph node cytotoxicity and the development of specific serum inhibitors to continued growth and dissemination of the tumour are discussed.

Immune response to Mycobacterium tuberculosis in rats.

After intravenous injection into rats, both the attenuated strain R1Rv and the virulent strain H37Rv of Mycobacterium tuberculosis grow in the liver and spleen. However, the infected rats mount a specific immune response with great rapidity, giving a false impression of natural resistance to the tubercle bacillus. Adoptive immunity to tuberculosis was achieved by transferring thoracic duct cells from immunized donors to normal syngeneic recipients. The transferred immunity was vested in a population of lymphocytes uncontaminated with macrophages. The adoptive immunity was effectively expressed against both attenuated and virulent tubercle bacilli, and it was shown to be immunologically specific. Lymphocytes which conferred immunity to tuberculosis were not protective against Listeria monocytogenes infection, and vice versa. Immunity could not be transferred with either normal thoracic duct lymphocytes (TDL), heat-killed sensitized TDL, or serum from specifically immunized donors. The ability of TDL from BCG-immunized donors to confer immunity was maintained at an unimpaired level for at least 3 months after immunization.

Increased cellular reaction to damage caused by angiotensin in arterioles of normal recipient rats after transfer of lymphocytes from hypertensive rats.

The presence of an immunological factor in vascular disease in renal hypertensive rats is demonstrated by showing, that transfer of thoracic duct cells from these rats to normal syngeneic recipients is followed by a change in the reactivity to injections of angiotensin with the result that the normal recipients react with a secondary cellular response in the damaged arterioles.