Strain differences in mouse cellular responses to Mycobacterium lepraemurium and BCG subcutaneous infections. I. Analysis of cell surface phenotype in local granulomas.

C57BL/6, BALB/c and CBA mice were subcutaneously infected with either Mycobacterium lepraemurium (MLM) or BCG, and studied for bacillary growth, granuloma size of infected footpads and draining lymph nodes (DLN), and DLN cell surface phenotype. Whereas, BCG-infected mice controlled the infection and developed early and large granulomas, MLM-infected mice exhibited major strain variations in their resistance to the infection, as well as in the granuloma size and kinetics. C57BL/6 mice, highly resistant, displayed early and regressive granulomas; BALB/c mice showed lower resistance and early granulomas that grew continuously; CBA mice, highly susceptible, developed late, soft, phagocyte-rich granulomas. Important strain differences in lymph node lymphocyte subset distribution could be observed prior to any infection: C57BL/6 mice displayed higher B cell percentages than both of the other strains and BALB/c mice showed the highest CD4/CD8 ratios, followed by CBA and C57BL/6 mice. BCG and MLM infections both induced similar changes of these parameters in all three strains: that is a decrease of the B cell percentage and a decrease of the CD4/CD8 ratio, and the strain differences observed in uninfected mice persisted. On the other hand, DLN cells stimulated by the infecting bacillus and interleukin 2 also displayed an increase of the CD8 T cell percentage as compared with normal lymph node cells, but this phenomenon was much less pronounced in BALB/c mice, whether infected by MLM or BCG, and in MLM-infected CBA mice, than in BCG- or MLM-infected C57BL/6 (B6) mice. Thus the ability of C57BL/6 mice to generate an early and persistent CD8 T cell response to mycobacteria may contribute to their resistance to MLM.     

Production of interleukin-1 by draining lymph node cells during the induction phase of contact sensitization in mice.

Biologically active interleukin-1 (IL-1) has been detected in supernatants of draining lymph node cells isolated from contact-sensitized mice. Induction of IL-1 was dependent upon the concentration of sensitizer applied and occurred within 2 hr of exposure. The IL-1 activity could not be attributed to other interleukins and was neutralized by a specific antiserum. Reduced concentrations of IL-1 were produced by cells isolated from the draining nodes of mice that had been pre-exposed to the sensitizer. Since IL-1 has the potential to influence several aspects of lymphocyte activation, the production of significant concentrations of biologically active IL-1 by draining lymph node cells indicates that it is likely to play an important role in the afferent phase of contact sensitization.     

The immune response to sheep erythrocytes in the mouse: II. A study of the cytological events in the draining lymph node utilizing cellular imprints

The cytological events of the primary and secondary immune response in the popliteal lymph node of Swiss White mice were studied following administration of sheep erythrocytes into the hind footpad. Four morphological features of cellular activity of immunologically competent cells—basophilia, synthesis of RNA, mitotic activity and distinctive cellular morphology—were analysed, and correlated with previous studies of 19S and 7S antibody forming cellular activity employing plaque assays performed on the residual lymphoid tissue remaining after production of node imprints.

The findings support the view that 19S and 7S antibody forming cells in the primary immune response are derived from two populations of cellular precursors. It is suggested that the lymphoid cell producing 19S immunoglobulin arises by transformation from the reticular cell following activation by antigen, while the 7S antibody forming cell arises from the small lymphocyte following some degree of initial transformation and subsequent cellular proliferation. The possibility that the 7S antibody forming cells had passed through a transient period of biosynthesis of 19S antibody was suggested in the present studies. Finally, evidence was provided for the presence of two morphological types of plasma cells, which, by virtue of their appearance at different stages of the primary immune response, could represent cells producing different immunoglobulins at varying rates of protein biosynthesis.

     

Interleukin-2 responses of MRL/lpr mouse splenocytes and lymph node cells induced by TPA and A23187

Treatment of splenocytes and lymph node cells of 5 month-old MRL/lpr mice with TPA induced IL-2-dependent proliferation of the cells in the presence of CA++. The induced response was inhibited completely by a monoclonal antibody to IL-2 receptor. The combination of TPA and A23187 in the lpr cells induced both proliferation and production of IL-2 in a Ca++-dependent fashion. The proliferative response of the lpr cells was equivalent to that of congenic (MRL/+/+) or normal cells, but the quantity of IL-2 secreted from the lpr cells was significantly less than that of the controls. Actinomycin D, but not mitomycin C, blocked IL-2 secretion from the treated lpr cells indicating de novo synthesis of IL-2 mRNA in the cells. Thus the lpr lymphocytes can be activated to proliferate in response to IL-2, yet they do not secrete IL-2 at a normal level even if activation signals are transmitted into the cells.     

Microplate culture of mouse lymph node cells. I. Quantitation of responses to allogeneic lymphocytes endotoxin and phytomitogens

A microplate culture system has been used to standardise mouse lymph node lymphocyte responses to concanavalin A (Con A), phytohaemagglutinin (PHA), pokeweed mitogen (PMW), endotoxin (LPS) and allogeneic lymphocytes and optimum culture and [³H]thymidine (³H-Tdr) labelling conditions determined.The effects on lymphocyte transformation of microplate well-shape, foetal calf serum, cell concentration, mitogen dose, culture time, buffering system and initial pH of culture were examined. These factors showed different effects, both quantitative and qualitative, on the kinetics of the responses to the various stimulants resulting in dissimilar optimal culture conditions. This probably reflects either the different subpopulations of lymphocytes activated by these stimulants, or the different modes of action of the stimulants.Optimal ³H-Tdr labelling conditions were achieved with saturation concentrations of exogenous Tdr of at least 3.0 μg/ml during culture in the most highly proliferating cultures. At saturation concentrations, any specific activity (SA) of ³H-Tdr could be used for quantitation since lymphocytes in different states of activation were affected by the radiobiological effects to the same extent. However, it was calculated from the parabolic relationship of ³H-Tdr incorporation and SA that at saturation concentrations the maximal SA that should be used to provide the highest uptake is 1.3 Ci/mM for a 24 hr pulse.     

In vitro production of interleukin-4 and interferon-gamma by lymph node cells from BALB/c mice infested with nymphal Ixodes ricinus ticks.

In this study we compared the ability of lymphocytes taken from axillary and brachial lymph nodes of BALB/c mice that had been infested once three times with 15 nymphal Ixodes ricinus ticks, to produce interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) after in vitro stimulation with concanavalin A (Con A). They released high levels of IL-4 and low levels of IFN-gamma. An increase of IFN-gamma between the first and the third tick infestation was observed. Salivary gland extracts from female I. ricinus ticks induced specific in vitro proliferation of lymphocytes from infested mice. IL-4 production was correlated with the salivary gland extracts' ability to stimulate tick-specific lymphocyte proliferation. Its levels remained high from the first to the third infestation. IFN-gamma production was not necessarily associated with tick salivary gland antigen stimulation. In BALB/c mice, anti-tick immune response induction is regional and the contribution of other similar secondary lymphoid organs is negligible. Only cells from the lymph nodes which drained the tick-fixation site proliferated in vitro in the presence of tick antigens, and when stimulated with Con A produced IL-4 and IFN-gamma.     

Mechanisms underlying the depressed production of interleukin-2 in spleen and lymph node cell cultures of mice infected with Mycobacterium bovis BCG.

Mice were infected intravenously with 1.0 mg of Mycobacterium bovis BCG. At various times thereafter, spleen and peripheral lymph node cells were stimulated with concanavalin A for 18 to 20 h, and their capacity to produce interleukin-2 (IL-2) was evaluated by means of a T-cell blast proliferation technique. A depression of IL-2 production that was complete in the spleen but partial in lymph node cell cultures occurred at 2 to 3 weeks and persisted till weeks 8 to 10 after infection. No direct evidence was found for an active suppressor mechanism depressing in vitro the production of IL-2. In spleen cell cultures the suppression of IL-2 production would result from a functional defect of the IL-2-producing T-cell subset, whereas in lymph node cell cultures the depression mainly results from a relative lack of IL-2-producing cells caused by an accumulation of immunoglobulin-positive and "null" cells. Spleen cells from BCG-infected mice maintained their capacity to acquire IL-2 receptors when activated by concanavalin A.     

Immunity to Mycobacterium leprae infections in mice stimulated by M. leprae, BCG, and graft-versus-host reactions.

Infections of mice with Mycobacterium leprae in one rear foot pad immunized them against a second infection in the other rear foot pad. Purified bacilli harvested from the first infection also produced immuniy when injection into the foot pads of previously uninfected mice. Injections of BCG afforded similar protection, but had no adjuvant effect on M. leprae. M. duvali, a cultivable mycobacterium that is reported to be more closely related antigenically to M. leprae than BCG is, provided much less protection against M. leprae challenge than BCG did. Moreover, when M. duvali was mixed with BCG, it was not any more effective than BCG alone. Graft-versus-host reactions, induced by injections of parental spleen cells into F1 hybrids, provided no protection against M. tuberculosis and M. marinum challenge. They gave moderate protection against M. leprae in one experiment but not in another with a different schedule. Allogenic spleen cells had a protective effect when injected locally into the infected foot pad. The effect produced by these injections of spleen cells was a delay in the appearance of bacterial growth; however, there was no decrease in the rate of logarithmic growth when it did appear and no reduction in the eventual plateau level.     

Enhanced interferon-gamma (IFN) production by lymph node cells from autoimmune (MRL/1, MRL/n) mice.

Interferons (IFNs) have been implicated in the aetiopathogenesis of the autoimmune disease systemic lupus erythematosus (SLE). The ability of unstimulated and Sepharose-bound concanavalin A (ConA) stimulated spleen or lymph node (LN) cells from normal CBA/T6 mice and histocompatible autoimmune strains (MRL/1, MRL/n) of various ages to produce IFN-gamma was measured. Levels of IFN-gamma produced by ConA-stimulated spleen cells from CBA/T6, MRL/1 and MRL/n mice at 6 weeks of age were not statistically different (mean +/- SEM: 786 +/- 182, 1147 +/- 282, 1024 +/- 146 IU/ml, respectively). At this age only stimulated LN cells from MRL/l mice produced detectable IFN-gamma (538 +/- 44 IU/ml) and these levels remained constant up to 6 months. IFN production by stimulated LN cells from young MRL/n mice (66 +/- 21 IU/ml at 3 months, 44 +/- 41 IU/ml at 6 months) increased at 1 year (463 +/- 97 IU/ml) corresponding to the age of disease onset. The failure of stimulated CBA/T6 LN cells to produce IFN-gamma was not due to cell-cell suppression, defective IL-2 production or the generation of soluble inhibitors. Stimulated LN cells from other normal inbred (C57Bl/6, Balb/c, A/J) outbred and FI hybrid mouse strains (Swiss, [Swiss x Balb/c] F1, (CBA/T65 x C57Bl/6]FI) produced undetectable or low levels of IFN compared to MRL/1 and MRL/n mice. These results show that autoimmune mouse LNs generate more IFN compared to normal controls and that the increase in IFN levels (at least in MRL/n) corresponds to the age of disease onset.     

Transferrin binding and iron uptake by mouse lymph node cells during transformation in response to concanavalin A.

Mouse lymph node cells cultured with Concanavalin A (Con A) in serum-free medium containing 59Fe-transferrin took up 59Fe more rapidly than cells cultured without Con-A. Uptake of iron commenced rapidly and preceded the onset of DNA synthesis in stimulated cells. Total uptake of transferrin during culture was much lower than that of iron, indicating that cells could remove iron from transferrin. The released transferrin appeared to be undergraded. Lymphoblasts bound six times more transferrin per cell than small lymphocytes. Lymphocytes also took up iron from citrate and nitrilotriacetate complexes, and iron so acquired was not readily removed by desferrioxamine, indicating that it was not bound extracellularly.     

Nonspecific augmentation of lymph node T cells and I-E-independent selective deletion of VβbTl4+ T cells by Mtv-2 in the DDD mouse

DDD/1 (DDD) mice were characterized by marked paucity of T cells in lymph nodes (LN). In DDD-Mtv-2/Mtv-2 (DDD-Mtv-2) congenics, T cells were 4- to 18-fold increased depending on ages but B cells doubled at the most. Thymus weight also increased. In DDDfDDD-Mtv-2, DDD neonatally infected with Mtv-2-derived exogenous MMTV (MMTV-2), neither LN cells nor thymus weight increased. The V_β 5⁺ and V_β 8⁺ Tcell contents in LN were practically the same among three strains. The Mtv-2-induced expansion of LN Tcells was polyclonal and appeared indigenous to DDD mice. Both Mtv-2 and MMTV-2 induced progressive age-dependent deletion of V_β 14⁺CD4⁺ LN cells. Mtv-2 but not MMTV-2 caused deletion of V_β 14⁺CD8+ LN cells and mature V_β 14⁺CD4⁺ thymocytes. Thus, Mtv-2- and MMTV-2-induced V_β 14⁺ T cell deletion may reflect intrathymic and peripheral elimination, respectively. The absence of I-E gene expression in DDD indicates that V_β 14⁺ T cell deletion advances independently of I-E molecules in this experimental system.     

Specific and nonspecific antitumor immunity. III. Specific T lymphocyte-mediated cytolysis of P815 mastocytoma and SL2 lymphoma by draining lymph node cells from syngeneic tumor-bearing DBA/2J mice.

Tumor-specific cytolytic activity, as measured by the 51Cr release assay, has been demonstrated in the draining lymph node cells from DBA/2 mice bearing the syngeneic P815 mastocytoma or SL2 lymphoma. This lytic activity is mediated by cytolytic T lymphocytes (CTL), since cytotoxicity is eliminated by treatment of the effector cells with anti-Thy 1.2 (theta) serum plus complement but is enhanced or unaffected by anti-Thy 1.2 serum alone, antimouse immunoglobulin plus complement, normal or aggregated mouse immunoglobulin, or removal of adherent cells. The time course of the CTL response has been analyzed and is similar for both P815 and SL2, with a peak around Days 10 to 12 after tumor grafting. Detectable CTL activity then wanes despite continued antigenic stimulation from the growing tumor. The ability of the immunotherapeutic agent Corynebacterium parvum to augment such specific CTL responses is documented as one antitumor pathway by which this agent may act.     

MHC class II expression by Langerhans' cells and lymph node dendritic cells: possible evidence for maturation of Langerhans' cells following contact sensitization.

Following exposure of mice to contact sensitizing chemicals, dendritic cells (DC) rapidly accumulate in the draining lymph nodes. A proportion, at least, of the DC which arrive in the nodes bear significant amounts of antigen and are derived from epidermal Langerhans' cells (LC). It is of interest that although LC are relatively inefficient antigen-presenting cells, the antigen-bearing DC found within draining nodes are potent accessory cells and induce immune responses both in vitro and in vivo. Previous in vitro studies have shown that during culture in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF), LC are subject to a functional and phenotypic maturation characterized by the development of effective accessory cell function and elevated membrane Ia antigen expression. We have hypothesized previously that LC may undergo a similar maturation in vivo as they move to the draining lymph nodes following receipt of the stimulus to migrate. As maturation in vitro is accompanied by increased Ia, we have examined the expression of this molecule on epidermal LC and lymph node DC during the induction phase of contact sensitization. The data reported provide evidence that peripheral lymph node DC, irrespective of whether they are derived from draining or resting nodes, and irrespective of whether or not they bear antigen, express comparable high levels of Ia antigen. In contrast, compared with DC, freshly isolated LC have considerably less (on average five times less) Ia antigen. These results indicate that during migration from the skin to lymphoid tissue LC are subject to a phenotypic maturation, comparable with that observed in vitro, and consistent with the acquisition of active antigen-presenting cell function.     

Development of antigen-induced proliferative responsiveness by murine lymph node cells. I. Identification of differences in the in vitro proliferative responses during a first and a second period of responsiveness.

The development and course of antigen-induced proliferative responsiveness by murine lymph node cells (LNC) was observed for 16 weeks post-immunization. The initial phase of responsiveness was characterized by antigen-induced proliferative responsiveness in vitro which reached a maximum 3-5 weeks post-immunization and then declined to low levels by 6-8 weeks. Without injection of additional antigen, the initial phase of responsiveness was followed by the development of a second phase of antigen-induced proliferative responsiveness 10-12 weeks post-immunization. These findings suggest that the in vivo development of lymph node lymphocytes capable of a proliferative response to antigen is under some type of modulation which is maximal 6-8 weeks post-immunization. Early in the first phase the proliferative responses to higher concentrations of antigen peaked early in the culture period (days 3-4), whereas responses to the lower concentrations of antigen were optimal after 5-6 days of culture. During the latter half of the first phase, however, peak proliferative responses were made to all the concentrations of antigen on the same day of culture (day 6). In contrast, the responses detected at the beginning and throughout the second phase of responsiveness were characterized by maximum proliferation to all the concentrations of antigen late in the culture period (day 7). These results delineate the temporal requirements for maturation of antigen-induced proliferative responsiveness of murine LNC post-immunization and indicate the time interval when optimal responses may be detected.     

Differences in [3H]-thymidine uptake of lymph node cells stimulated by Con A and PHA in H-2 congenic mouse strains.

The genetic control of the lymphocyte responsiveness to Con A and PHA-P has been studied by using inbred, H-2 congenic mouse strains. Segregation studies were carried out on F1 and backcross mice of a high and a low responder strain. According to the results, there is a strong correlation between the responsiveness of lymph node cells to mitogens and H-2 haplotypes in different H-2 congenic strains of mice and in different backcross generations. High responsiveness to PHA-P is associated with H-2b, while low responsiveness with H-2a or H-2k haplotypes, but the correlation is the inverse in response to Con A, in the parental strains and backcross mice as well. The magnitude of the responses to both Con A and PHA-P was found to be intermediate in F1 heterozygotes of a low and high responder parental strain. The effect of other non H-2 genes on the responsiveness to these mitogens has also been demonstrated.     

Short lived, dividing cells mediate adoptive transfer of immunity to Trichinella spiralis in mice. I. Availability of cells in primary and secondary infections in relation to cellular changes in the mesenteric lymph node.

After a primary infection with the parasitic nematode Trichinella spiralis NIH mice showed a short lived increase in cellularity of the mesenteric lymph node (MLN), which began between days 2 and 4 peaked at day 8 and had declined by day 21. The majority of cells contributing to this increase were Ig-ve and presumed to be T cells. Coincident with the increase in cell number there was an increase in lymphoblast activity, again largely in the T-cell fraction. MLN cells taken at intervals from mice during a primary infection successfully transferred immunity, i.e. accelerated worm expulsion in recipients, on days 4 and 8, but not on day 21. It was shown that the effective cells in transferring immunity were present in the T-enriched fraction. When mice were present in the T-enriched fraction 21 days after a primary infection the same sequence of changes was apparent in the MLN, but the time course was accelerated, i.e. peak cellularity and lymphoblast activity occurred on day 4 post challenge. Cells capable of transferring immunity were present in the MLN on days 2 and 4 post challenge but not thereafter. As in the primary infection the effective cells, and those responsible for the cellular changes in the MLN, were T cells.     

Radioautographic study of cellular mechanisms in delayed hypersensitivity: IV. Distribution of injected lymph node, spleen, thymus and bone marrow cells

Mononuclear cells from various organs of sensitized donor rats were labelled in vitro with tritiated leucine and injected intravenously into sensitized, syngeneic recipients. The localization of injected cells was studied in radioautographs.

Bone marrow cells were the most frequent of the labelled cells in skin reactions. Equal numbers of bone marrow cells were found in specific and non-specific delayed reactions and irritation reactions induced with turpentine.

Lymph node cells were found in delayed but not in turpentine reactions. Spleen cells were less frequent than lymph node cells in delayed skin reactions; small numbers were found in the turpentine reactions. Lymph node and spleen cells did not show detectable antigenic specificity.

Thymus cells were not found in the skin sites, even when they were allowed to circulate 2–3 days in the recipients before biopsies were taken.

Irrespective of the source of the cells injected few or no labelled cells were found in the recipient thymuses. Lymph node and spleen cells migrated equally into lymph nodes and spleen, but were infrequent in bone marrow. Only bone marrow cells had a decided preference for their organ of origin.

     

Activation of CD4+ and CD8+ lymphocyte subsets by streptozotocin in the popliteal lymph node assay. II. Comparison with acute graft-vs-host reaction in H-2 incompatible F1 mouse hybrids.

Changes in lymphocyte subsets during an acute GVH reaction were compared to STZ-induced PLN response in mice. The GVH reaction was induced locally by sc injection of parental C57Bl/6 [B6] spleen cells into (C57Bl/6 x DBA/2) F1 footpad [B6D2F1]. Early cell activation and time-related changes in T- and B-lymphocyte subsets were monitored during the onset of the GVH reaction by flow cytometry and immunophenotyping. Examination of cell size and chromatin decondensing for T- and B-cell subsets showed differences in activation profile during the early phase of the GVH reaction. The present study provides direct evidence for early in vivo activation of both CD4+ and CD8+ T-cells. Our data confirm the central role of T-cell activation in the induction of a GVH reaction and suggest that recirculatory host B-cells can play an important role in early GVH node enlargement. Overall, our comparative analysis supports the concept of polyclonal T-cell activation for both STZ-related and GVH-induced lymphoproliferation. Chemicals-induced lymphoproliferation leading to autoimmune reactions is a challenging issue. A number of drugs and chemicals have been tested in the PLNA assay for lymphoproliferative potential. We previously reported the activation and proliferation of T-cell subsets following STZ injection into murine footpads. The STZ-induced PLN enlargement and proliferation characteristics of T- and B-cell subsets were postulated to be similar to those of an acute allogeneic GVHR. In the present study, a cytometric analysis of T- and B-cell subsets in PLNs was performed during an acute allogeneic GVHR, for comparison purposes. Such a reaction results in a massive node enlargement five to ten times that seen after stimulation with conventional antigens. Acute GVHR is believed to be a direct consequence of the high frequency of alloreactive donor T-cells inducing a massive proliferation of B-cells, almost exclusively of host origin, in GVHR nodes. It is now widely accepted that donor T-cells activated as the result of exposure to foreign MHC antigens in the recipient, secrete various cytokines which assist the host B-cells and bypass the normal B-T cell cooperation. Induction of an acute GVHR, as in the parental B6--->recipient B6D2F1 model, requires the injection of CD4+ and CD8+ donor T-cells into an F1 recipient that differs from the parent at both MHC class I and II loci.(ABSTRACT TRUNCATED AT 400 WORDS)     

The primary responses of murine neonatal lymph node CD4+ cells are Th2-skewed and are sufficient for the development of Th2-biased memory

Exposure of neonatal mice to antigen often results in Th2-biased responses in later life. Examples of this Th2 tendency are (a) secondary antibody responses dominated by the Th2-associated IgG1 isotype and (b) Th2-mediated tolerance to alloantigens. We previously reported that neonates develop primary Th1 and Th2 function in the lymph nodes but exclusive Th2 primary splenic responses. Here, we have tested whether the Th2 bias of adults initially immunized as neonates is due to the early, primary Th2 polarization in the spleen. Surprisingly, removal of the spleen at birth had no affect on either IgG1-dominant secondary responses or the development of tolerance to alloantigens. Thus, neonatal lymph nodes are sufficient to generate Th2-biased function following neonatal antigen exposure. To understand how this could arise, we examined the primary Th1/Th2 responses of CD4+ lymph node cells. Unlike the balanced Th1/Th2 responses seen with total lymph node cells, the primary responses of isolated CD4+ cells were skewed to IL-4 producing function. These results suggest that the early development of Th2-dominant responses by lymph node CD4+ cells contributes substantially to the subsequent development of Th2-dominant memory in neonates.