Responses of the thymus and the paracortex of draining lymph nodes to repeated applications of oxazolone to mouse skin.

The oxazolone-induced response in the paracortex of draining lymph nodes is characterized by an early increase in the proliferative activity that decreases to control levels when stimulation is continued. The possibility that this may be a toxic side effect of the concentrated oxazolone solution used was investigated by simultaneous registration of the changes taking place in the thymus. These were found to be different from toxin-induced changes and compatible with cell loss due to massive emigration of cells. Repopulation of the thymus took place over the last 1 1/2 week of stimulation. It was concluded that the changes in the thymus as well as the decline of the proliferative activity in the paracortex, are most likely physiological responses. The most important factor in maintaining a high production of paracortical lymphocytes under chronic stimulation is the increase in the lymphocyte mass in the paracortex.     

Regeneration of autotransplanted lymph node fragments

Summary In normal young minipigs thin slices of autologous mesenteric or superficial inguinal lymph nodes were implanted either in the greater omentum or subcutaneously in the groin region. The regeneration was studied histologically and connections between the afferent lymphatics and the regenerated tissue were checked. In the greater omentum, no regenerated lymph node tissue was found. In the inguinal region, lymphoid tissue with all the typical lymph node compartments was identified following antigenic stimulation in the draining area. Sinuses, germinal centres with a lymphatic corona, and a paracortex with typical high endothelial venules were seen. There was evidence of afferent lymphatics, e.g., macroscopically visible lymphatics, the occurrence of a subcutaneously injected dye, the effect of antigenic stimulation and a normal lymph node structure. Avascular transplants of autologous lymph node fragments regenerate subcutaneously, possibly providing a future technique for treating lymphoedema after radical excision or irradiation of lymph nodes.     

Lymph node accessory cells in the immune response

Summary The immune response in the rat parathymic lymph node was studied after administration of antigen into the peritoneal cavity. Special attention was paid to the accessory cells,which might induce the response. During the induction phase of the response a heterogeneous population of non-lymphoid mononuclear cells was present in the subcapsular sinus and the cortex of the node. These cells resemble veiled cells described in skin draining lymph and interdigitating cells in the paracortex of skin draining lymph nodes, but they do not contain Birbeck granules. It is concluded that the appearance of these granules depends on the site of the exudate provocation and that the presence of the organelles in these accessory cells is not obligatory for lymphocyte stimulation.     

Dynamic and morphological responses of draining lymph nodes to single and repeated applications of oxazolone to guinea-pig skin

Auricular lymph nodes in guinea-pigs were studied for 3 weeks under continuous stimulation with oxazolone applied to the ear skin. Quantitation of 3H-Tdr labelled paracortical lymphocytes following pulse labeling, demonstrated a marked, but only transitory rise in the proportion of cells in DNA synthesis. In spite of this, the total number of cells in S-phase continued to rise during the remaining part of the observation time, as a result of a steady increase of the paracortical cell mass. The variation in the proportion of large, pyroninophilic blast cells, revealed a pattern similar to that of the labelling index. A high proportion of blast cells was found only in the initial phase of the reaction, while the chronic response was characterized by a low proportion, no different from the starting level. Paracortical enlargement appeared to be the most reliable morphological criterion on which a chronically stimulated paracortex could be distinguished from an "unstimulated". The initial paracortical response, similar to a primary reaction, was followed by an equally pronounced development of germinal centres and plasma cells. These were also persistent features under continued stimulation. The reported changes are most likely specific responses to oxazolone stimulation.     

Cell-mediated immune response after vasectomy in rats.

This study investigated the development of a cell-mediated immune response after vasectomy in Swiss Albino rats, by comparing the development of the thymus-dependent lymphoid tissue of the regional testicular lymph node and the spleen in vasectomized and in sham-operated control animals. Frozen sections were used and thymus-dependent regions were stained by immunocytochemistry. After vasectomy, the areas occupied by the paracortex in the lymph node sections showed a significant increase in size; the thymus-dependent regions of the spleen, in contrast, showed no change. The regional lymph node, rather than the spleen, seems to be important in cell-mediated and humoral immune responses to vasectomy.     

Guinea pigs immunized with xenogeneic cells: skin test, in vitro stimulation; cytotoxicity of lymph node cells

Guinea pigs were immunized with cells of a human lymphoblastoid line. While nonsensitized lymph node cells were not influenced, those derived from immunized animals were markedly stimulated in vitro by the human cells. Maximal activity was found with lymphocytes harvested on day 6. The response was paralleled with the strength of skin induration elicited by the human cells and with the efficiency of the cytotoxic effect of lymphocytes on human target cells. The stimulation and cytotoxicity was specific while the skin test demonstrated cross reactivity with mouse cells.     

On-Lattice Simulation of T Cell Motility,Chemotaxis, and Trafficking in the Lymph Node Paracortex

Agent-based simulation is a powerful method for investigating the complex interplay of the processes occurring in a lymph node during an adaptive immune response. We have previously established an agent-based modeling framework for the interactions between T cells and dendritic cells within the paracortex of lymph nodes. This model simulates in three dimensions the “random-walk” T cell motility observed in vivo, so that cells interact in space and time as they process signals and commit to action such as proliferation. On-lattice treatment of cell motility allows large numbers of densely packed cells to be simulated, so that the low frequency of T cells capable of responding to a single antigen can be dealt with realistically. In this paper we build on this model by incorporating new numerical methods to address the crucial processes of T cell ingress and egress, and chemotaxis, within the lymph node. These methods enable simulation of the dramatic expansion and contraction of the T cell population in the lymph node paracortex during an immune response. They also provide a novel probabilistic method to simulate chemotaxis that will be generally useful in simulating other biological processes in which chemotaxis is an important feature.     

The effects of intravenous administration of Methanol Extraction Residue (MER) of tubercle bacilli in the dog

Summary The effects of intravenous administration of the insoluble methanol extraction residue (MER) of tubercle bacilli were studied in six adult Beagle dogs. All animals were closely monitored for clinical signs, alterations in serum biochemistry or hematologic values, and for gross and histologic findings at autopsy. Immune responsiveness was assessed by skin reactivity to test antigens and by ability of spleen and lymph node lymphocytes to produce macrophage activating factor (MAF).Regardless of the dose of MER or frequency of administration, there were no clinical or biochemical alterations detected in the animals. Immediately after injection of MER, a massive but transient granulocytopenia appeared. Skin reactivity failed to demonstrate any alterations in immune responsiveness, but lymphocytes from MER-treated dogs released MAF in response to in vitro stimulation with purified protein derivative (PPD) in contrast to those from nontreated control dogs.Histologic findings were associated with the immune response to MER and were most dramatic in the liver and lungs, where formation of a large number of epithelioid granulomas was observed. MER, intravenously administered in relatively high doses, was well tolerated by dogs, in whom it produced little evidence of toxicity, and seemed to increase some systemic immunologic effects.     

Stochastic regulation of cell migration from the efferent lymph to oxazolone-stimulated skin

The systemic immune response is a dynamic process involving the trafficking of lymphocytes from the Ag-stimulated lymph node to the peripheral tissue. Studies in sheep have demonstrated several phases of cell output in the efferent lymph after Ag stimulation. When skin contact sensitizers are used as Ag, the efferent lymph cell output peaks approximately 96 h after Ag stimulation and is temporally associated with the recruitment of cells into the skin. To investigate the relative contribution of this high-output phase of efferent lymphocytes to lymphocytic inflammation in the skin, we used a common contact sensitizer 2-phenyl-4-ethoxymethylene-5-oxazolone (oxazolone) to stimulate the skin and draining prescapular lymph node of adult sheep. The efferent lymph ducts draining the Ag-stimulated and contralateral control lymph nodes were cannulated throughout the experimental period. The lymphocytes leaving the lymph nodes during the 72-h period before maximum infiltration were differentially labeled with fluorescent tracers, reinjected into the arterial circulation, and tracked to the site of Ag stimulation. Quantitative tissue cytometry of the skin at the conclusion of the injection period (96 h after Ag stimulation) demonstrated more migratory cells derived from the Ag-stimulated lymph node than the contralateral control (median 18.5 vs 15.5 per field; p < 0.05). However, when corrected for total cell output of the lymph node, the Ag-stimulated migratory cells were 3.8-fold more prevalent in the skin than the contralateral control cells. These results suggest that the in situ immune response generally mirrors the frequency of recruitable lymphocytes in the peripheral blood.     

Donor-specific and nonspecific impairment of graft-versus-host reactivity in F1 hybrid rats after pretreatment with parental strain lymphocytes.

The relative contribution of donor-specific and nonspecific impairment of graft-versus-host (GVH) reactivity in F₁ hybrid rats pretreated with graded doses of parental strain lymphocytes was investigated, over a wide dose range, using the popliteal lymph node assay. Donor-specific and nonspecific impairment were shown to follow completely different dose/response curves peaking at widely different pretreatment doses. After pretreatment with certain doses of parental strain lymphocytes donor-specific and nonspecific impairment were induced simultaneously and the impairment of GVH reactivity in response to the pretreatment strain then appeared to be due to the combined effect of both. The possibility that donor-specific and nonspecific impairment might be induced by different lymphocyte populations in the pretreatment inoculum is discussed.     

The ultrastructure of the abdominal lymph nodes cortex in rats during primary and secondary immune response (a special reference to dendritic reticulum cells and interdigitating cells)

The stimulation of coeliac rat lymph nodes was performed by intraperitoneal injections of typhoid vaccine and was unique for the primary immune response and repeated after 6 weeks for the secondary response. The light and electron microscopic observations showed for the primary response, an early germinal center reaction, which might be accounted for by a background of continuous stimulation of the coeliac nodes, stemming from the digestive tract. The dendritic reticulum cells (DRC), considered typical for the B area, were located at the borderline between the germinal center and the mantle zone. Their cytoplasmic extensions penetrated the lymphocyte-lymphoblastic center, surrounding most of the germinal center cells. The marginal zone and the paracortex reacted as a whole, the interdigitating cells (IDC) being the dominant feature. An explanation would be that the marginal zone can be penetrated by T cells and connected IDCs, thus, the B and T areas seem to be largely interspersed. The results suggest that IDCs are cells of direct monocytic origin.     

Immunohistochemical analysis of human peripheral blood and lymphoid tissues using monoclonal antibodies immunoreactive with non-lymphoid cells

Immunoperoxidase methods were used to study human peripheral blood and lymphoid tissues using a panel of monoclonal antibodies to non-lymphoid cells. The majority of peripheral blood monocytes were immunoreactive for LeuM1, LeuM2, LeuM3 and LeuM5. Rare peripheral blood monocytes were immunoreactive for R4/23. The different antibodies showed characteristic patterns of immunoreactivity in peripheral lymphoid tissues. LeuM1 was immunoreactive with scattered cells in the paracortex of lymph node and tonsil and with many cells in the marginal zone of the spleen. LeuM2 was immunoreactive with endothelial cells in lymph node and tonsil. A few cells in the red pulp of the spleen were immunoreactive for LeuM2. LeuM3 and R4/23 showed distinctive immunoreactivity in germinal centers of secondary follicles, giving a "lacy" pattern. LeuM3 was also immunoreactive with endothelium in lymph node and tonsil and with sinus lining cells in lymph node. LeuM5 was immunoreactive with macrophages in the germinal center, fibroblastic reticulum cells in the mantle zone and interdigitating reticulum cells in the paracortex of lymph node and tonsil.     

The role of dendritic cells in the initiation of immune responses to contact sensitizers. I. In vivo exposure to antigen

Twenty-four hours after skin painting mice with picryl chloride (PIC) there was a four- to fivefold increase in the numbers of dendritic cells (DC) isolated from the lymph nodes. These DC initiated primary proliferative and cytotoxic responses when added to cultures of normal syngeneic lymph node cells. The proliferative response was enhanced when the donors of the responding lymph node cells were sensitized with the same antigen. Contact sensitivity developed in syngeneic mice injected into the footpads with 30,000-50,000 DC from lymph nodes of mice painted with picryl chloride 1 day previously. Thus, 1 day after skin painting mice, there were dendritic cells in the draining lymph nodes which were able both to initiate primary stimulation of lymphocytes in vitro and to sensitize recipient mice to give specific delayed hypersensitivity reactions.     

Delayed hypersensitivity in mice infected with reovirus. I. Identification of host and viral gene products responsible for the immune response

Delayed-type hypersensitivity (DTH) can be demonstrated in mice infected with reovirus by challenging primed animals in the footpad with virus. Maximal responses occur 7 days after immunization with as little as 10(5) viral particles. DTH to reovirus is transferable by lymph node cells and is mediated by T cells as the transfer of reactivity can be abrogated by treatment of cells with anti-Thy 1.2 plus complement. DTH to reovirus is serotype specific, animals infected with reovirus type 1 or 3 only develop DTH responses when challenged with the same serotype with which they were infected. Using recombinant viral clones containing genes from both parental serotypes, we have demonstrated that the S1 gene, the gene encoding the viral hemagglutinin, determines serotype specificity. Furthermore, in adoptive transfer experiments between mice of varying histocompatibility backgrounds, it was found that D or K, IA-IB region identity was required for the transfer of reactivity. These studies demonstrate that specific host and viral genes determine the in vivo cellular immune response to reovirus and should allow a more precise definition of the host cellular immune response to viral antigens.     

Acute SIV Infection in Sooty Mangabey Monkeys Is Characterized by Rapid Virus Clearance from Lymph Nodes and Absence of Productive Infection in Germinal Centers

Lymphoid tissue immunopathology is a characteristic feature of chronic HIV/SIV infection in AIDS-susceptible species, but is absent in SIV-infected natural hosts. To investigate factors contributing to this difference, we compared germinal center development and SIV RNA distribution in peripheral lymph nodes during primary SIV infection of the natural host sooty mangabey and the non-natural host pig-tailed macaque. Although SIV-infected cells were detected in the lymph node of both species at two weeks post infection, they were confined to the lymph node paracortex in immune-competent mangabeys but were seen in both the paracortex and the germinal center of SIV-infected macaques. By six weeks post infection, SIV-infected cells were no longer detected in the lymph node of sooty mangabeys. The difference in localization and rate of disappearance of SIV-infected cells between the two species was associated with trapping of cell-free virus on follicular dendritic cells and higher numbers of germinal center CD4⁺ T lymphocytes in macaques post SIV infection. Our data suggests that fundamental differences in the germinal center microenvironment prevent productive SIV infection within the lymph node germinal centers of natural hosts contributing to sustained immune competency.     

Inhibition of delayed hypersensitivity reactions in mice by colchicine. I. Mechanism of inhibition of contact sensitivity in vivo

Colchicine has been recently shown to inhibit delayed hypersensitivity reactions (DHR). In the present study we investigated the effects of colchicine on contact sensitivity (CS) to dinitrofluorobenzene. Colchicine, at a dosage level of 15 micrograms/mouse, inhibited the elicitation of the contact response only when given on the day of ear challenge. Administration of the drug during the induction phase did not have any effect on the CS reaction. By using adoptive transfer experiments, we could demonstrate that CS was suppressed only when colchicine was given to the recipient mice, while treating the donors of immune lymph node cells (I-LNC) did not affect their ability to transfer a significant DHR. These findings were observed also when I-LNC were directly injected into the ears, a result which indicated that there was no effect of the drug on the ability of effector cells to migrate to the site of antigen challenge. Neither was there any effect on the distribution of T cell subsets in peripheral lymph nodes. The proliferative response of LNC to antigenic or mitogenic stimulation in vivo or in vitro was also not affected by colchicine pretreatment. These findings raise major questions about the mechanism of action of colchicine in vivo and suggest that more experimentation is required to probe the mechanism of colchicine-induced suppression of DHR.     

Changes in dendritic cell migration and activation during SIV infection suggest a role in initial viral spread and eventual immunosuppression

Dendritic cells (DC) serve an essential function in linking the innate and acquired immune responses to antigen. Peripheral DC acquire antigen and migrate to draining lymph nodes, where they localize to the T cell-rich paracortex and function as potent antigen presenting cells. We examined the effects of human immunodeficiency virus (HIV) infection on DC function in vivo using the rhesus macaque/simian immunodeficiency virus (SIV) model. Our data show that during acute SIV infection, Langerhans cell density is reduced in skin and activated DC are increased in proportion in lymph nodes, whereas during AIDS, DC migration from skin and activation within lymph nodes are suppressed. These findings suggest that changes in DC function at different times during the course of infection may serve to promote virus dissemination and persistence: early during infection, DC mobilization may facilitate virus spread to susceptible lymph node T cell populations, whereas depressed DC function during advanced infection could promote generalized immunosuppression.     

Enhancement of the mixed lymphocyte response in the mouse by addition of thymocytes

Synthesis of DNA by mixtures of mouse lymph node and thymic cells was studied in vitro using mitomycin-treated allogeneic spleen cells as stimulator cells. The tests were performed to see whether there occurs a similar cell synergy during this reaction as has been reported during the in vivo graft-vs-host response.It was observed that mixtures of thymocytes and lymph node cells give higher incorporations of isotope-labelled thymidine than can be explained by a pure additive effect of the two cell populations tested separately. This enhancement of the reactivity was more pronounced using combinations of lymph node cells and medullary thymocytes obtained from cortisone-treated donors. Enhancement was also noted between lymph node cells and spleen cells. Blocking of the capacity of lymph node cells to synthesize DNA by treatment with mitomycin abolished this enhanced activity when mixed with thymic cells. On the contrary, mitomycin treatment of thymocytes did not abolish their capacity to increase the reactivity when mixed with normal lymph node cells. Thymocytes, which were unresponsive to the mitomycin-treated cells for genetic reasons, were also found to increase DNA synthesis when combined with lymph node cells. The mechanism by which thymocytes increase DNA synthesis of lymph node cells is not clear, but the results show that they have to be present during the reaction, since culture medium “conditioned” by thymocytes did not exhibit any enhanced capacity to promote a mixed lymphocyte reaction of lymph node cells.The results are thus in agreement with the findings obtained by others showing that mixtures of lymph node cells and thymic cells yield higher immunological reactivities in vivo against foreign transplantation, antigens than can be explained by a pure additive effect of the reactivities by the two cell populations tested separately. However, in contrast to these findings, the thymic cells do not have to be able to synthesize DNA or to react against the foreign cells in vitro to yield an enhanced response when mixed with lymph node cells.     

Cell surface glycoproteins of rat lymphocytes. I. Correlation of mitogenic stimulation by periodate or neuraminidase and galactose oxidase with the presence of papain-sensitive glycoproteins.

Oxidation of viable rat lymph node lymphocytes with either periodate or a combination of neuraminidase and galactose oxidase (NGO), followed by reduction with tritiated sodium borohydride, labels similar sets of cell-surface molecules as assessed by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Periodate and NGO induce blast transformation of lymph node lymphocytes (oxidative mitogenesis), and borohydride reduction inhibits the proliferative response. Thus, it is inferred that some or all of the glycoproteins that are labeled with tritiated borohydride may be involved in mediating the stimulation caused by the oxidizing agents. Treatment of lymph node lymphocytes with 5 units/ml papain abolishes the response to periodate or NGO but does not significantly affect the response to Con A. At the same time, papain treatment eliminates the labeled bands representing six high m.w. glycoproteins (175,000, 170,000, 160,000, 155,000, 100,000, and 70,000 daltons). No significant effect is seen on the labeling of the other components visualized in the slab gels. The results implicate the subset of six high m.w. papain-sensitive sialoglycoproteins in mediating oxidative mitogenesis of rat lymph node lymphocytes.     

Inhibition of erythroid cell growth by allogeneic murine lymphocytes. Evidence for a synergism between lymph node cells and thymocytes

Murine lymphoid cells from thymus and lymph nodes were tested for synergistic response in a graft-vs-host test. The test is based on the principle that allogeneic lymphocytes inhibit erythroid cell proliferation in the spleens of irradiated mice infused with syngeneic bone marrow cells.I was observed that mixtures of thymocytes and lymph node cells from the same parental strain yielded graft-vs-host responses in irradiated F₁-hybrids higher than expected by summing the responses of the two cell populations tested separately. A similar synergistic response was obtained using mixtures of thymocytes and lymph node cells obtained from the two parental strains of the hybrid, whereas such an effect was not detected using mixtures of lymph node cells or mixtures of thymocytes from the two parental strains. Nor could synergy be demonstrated between parental strain lymph node cells and thymocytes syngeneic with the bone marrow target cells. Thymocytes obtained from one parental strain which were injected into its irradiated F₁-hybrid transformed into a population of sensitized cells in the spleens of the recipients. This transformation was suppressed by the simultaneous injection of lymph node cells from the second parental strain. Since there is a synergistic immune response by such cell mixtures it is concluded that thymocytes may enhance the graft-vs-host response of lymph node cells. Parental strain thymocytes and lymph node cells, the latter being specifically immunologically tolerant to the bone marrow target cells, failed to give a synergistic response indicating that thymocytes do not transform unresponsive lymphocytes into responsive, but rather enhance the reactivity of existing, specifically responsive cells.The results thus show that thymocytes may enhance the response of lymph node cells in this specific graft-vs-host assay.