Relation between Enhanced Antibody Responses Elicited by Adjuvant Injection and Those Elicited by Secondary Antigenic Stimulation

An attempt was made to determine if there is any common mechanism in the enhanced antibody response caused either by injection of adjuvant, such as bacterial endotoxin (LPS) and complexed polynucleotides, or by secondary antigenic stimulation. LPS inoculated in mice 4 days before injection of sheep red blood cells (SRBC) and polyA:U invalidated the adjuvant effect of polyA:U injected together with SRBC, and the hemolysin plaque-forming cell (PFC) response of such mice was similar to that of the mice which received SRBC alone. When mice primed with SRBC 24 days in advance were injected with LPS and 4 days later re-stimulated with SRBC, their PFC response to the secondary stimulation was suppressed to less than one tenth of the normal secondary PFC response. The suppressive effect of LPS on the secondary antibody response was abolished if the serum collected from mice injected with LPS was given to the primed and LPS-injected mice at the time of the secondary antigenic stimulation. From these results we discussed the possibility that some common mediator might play a role in the enhanced antibody response elicited by either adjuvant injection or secondary injection of antigen.     

Peritoneal macrophages introduced into mouse foot pads enter the germinal center of regional lymph nodes nonspecifically.

Male mice were injected into their foot pads with sheep erythrocytes (SRBC) to form lymph follicles in the germinal centers in the popliteal lymph nodes. 4 weeks later, peritoneal macrophages labeled with carbon from syngeneic donors sensitized with SRBC or typhoid-paratyphoid bacilli (TAB) were separately injected into the foot pads as well. The popliteal lymph nodes were histologically examined at 6 h to 5 days after injection. Labeled macrophages appeared in the marginal sinus, migrated straight across the cortex from the marginal sinus to the lymph follicles and then entered the germinal centers. There was no difference in the mode of appearance, migration and localization of labeled macrophages in the regional lymph nodes between the mice given labeled macrophages from SRBC-sensitized donors and those given macrophages from TAB-sensitized donors. The entrance of lymph macrophages into the germinal centers of the regional lymph nodes would be immunologically nonspecific. After the injection of Pelikan ink into the foot pads, the macrophages which have taken up carbon in the peripheral tissue reached the regional lymph nodes via the afferent lymphatics and then entered the germinal centers, mainly through the medullary pole of the lymph follicles, after migrating along their immediate exterior from their marginal sinus to their medullary pole.     

Effects of Cholera Toxin on Delayed-Type Hypersensitivity to Sheep Red Blood Cells Inoculated Intranasally into Mice

The effects of cholera toxin (CT) on delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) were studied in mice sensitized by intranasal administration of SRBC. CT (1 μg/mouse), given intranasally together with SRBC (2 × 10⁷/mouse), induced a maximally enhanced DTH response, which reached its peak around 7 days after sensitization, and also induced an accelerated DTH response upon a second administration of SRBC 28 days later. The ability of CT to enhance the DTH to SRBC was lost, either when CT was administered via the intraperitoneal or subcutaneous route, or when CT was introduced into the nasal site from which a large proportion of the SRBC was discharged 2 days after SRBC administration. These results indicate that the cells that are located in the nasal site and participate in the earlier events of DTH response were most affected by CT. The following effects of CT on the earlier events, which occur within 24 hr after the intranasal administration of both CT and SRBC, appeared to be involved in the mechanisms by which CT enhances DTH to SRBC: (i) facilitation of the penetration of the antigen into the nasal tissue; (ii) reinforcement of the migration of immunocompetent cells from the blood to the nasal tissues; (iii) promotion of the ability of Ia-positive macrophages to present the antigenic determinants to T cells; (iv) facilitation of the differentiation of primed T cells to DTH-effector T cells.     

Distribution and morphologic characteristics of lymphoid cells in the canine popliteal lymph nodes in their normal state and under antigen effect

By means of morphometry, light and electron microscopy methods peculiarities in distribution of small, middle and large lymphocytes, as well as plasmocytes in various zones of the popliteal lymph nodes have been studied in normal and in dynamics up to one year after subcutaneous injection of BCG vaccine into the left hind paw. The antigen produces certain changes in density and morphological parameters of lymphoid cells both in the regional and in the contralateral lymph nodes. For them 3 periods are specific. During the first 3 days they are not antigen-dependent (stipulated by the stress reaction), during 7-24 days antigen-dependent processes of proliferation and differentiation of lymphocytes get into action. In 3 months a new wave of the immune response is observed.     

Alterations in antigen-induced DNA synthesis by specifically localizing cells and other lymphoid cells as a function of immunological memory

The effect of prior immunological experience on the antigen-induced DNA synthesis by specifically localizing cells (SLC) and other cells capable of migrating into lymph nodes (nonspecific cells, NSC) has been studied. Prior immunization with a homologous red cell antigen (sheep red blood cells, SRBC) results in enhanced DNA synthesis, relative to controls pretreated with saline, by NSC but not SLC for the first 48 hr following secondary immunization. By 72 hr both SLC and NSC show markedly reduced DNA synthesis, the SLC showing the greater reduction. Prior immunization with the strongly cross-reacting antigen, ox red blood cells (ORBC), shows a similar but milder effect; prior immunization with the non-cross-reacting antigen chicken red blood cells (CRBC) is without effect, though a similar depression of DNA synthesis by both SLC and NSC occurs 72 hr after secondary immunization with CRBC. In the presence of a concomitant secondary reaction to a non-cross-reacting (CRBC) or very weakly cross-reacting (horse red blood cells, HRBC) antigen, DNA syntheses by SLC-SRBC and NSC are depressed to approximately the same extent, suggesting that the greater depression of DNA synthesis by SLC seen in homologous secondary reactions reflects the additive effects of clonally specific and nonspecific suppressive factors. A comparison of the DNA synthesis by cross-reacting (ORBC) and non-cross-reacting (SRBC) SLC in mice given SRBC following preimmunization with ORBC suggests that “educated” SLC are considerably more resistant to the effects of nonspecific suppressors than “naive” SLC. Passively administered antibody, in concentrations that markedly reduced the numbers of direct plaque-forming cells appearing 72 hr after primary immunization, had no significant effect on DNA synthesis by SLC or NSC.     

Defective production of interleukin 1 and interleukin 2 in patients with systemic lupus erythematosus (SLE)

The effects of local antigenic exposure on the responsiveness of systemic T cells were evaluated after C3H mice were given drinking water containing 6% bovine serum albumin (BSA) for 10 days and challenged sc with 1.0 mg BSA in adjuvant 28 days after the initiation of antigen feeding. During the first 28 days, no evidence of in vitro antigen-induced proliferation [( 3H]thymidine incorporation) was detected in whole lymphocyte populations from the peripheral lymph nodes (PLN), spleen, or mesenteric nodes. In contrast, PLN cells treated with anti-Lyt-1 plus complement (C) had a significant proliferative response only if the cells were obtained during the first 6 days of antigen ingestion. Lymphoid cells from the same animals, treated with anti-Lyt-2 and C, did not respond to antigen. Two or 4 days after the injection, given on day 28, whole PLN cell populations from antigen-fed mice showed proliferation. No response was observed with PLN cells obtained 8 days after injection. Shortening the interval between the initiation of feeding and parenteral challenge partially restored proliferative responses detected 8 days after injection. Cultures prepared 4 days after simultaneous oral and parenteral antigenic exposure showed proliferation equal to or greater than cultures from mice that received only the injection. These data show that systemic T cell responsiveness is not eliminated by ingestion of soluble antigen, but rather is modulated in a manner previously detected in the humoral immune system.     

抗原物质引起滤泡的形成:一次投入与多次投入的比较

实验以不同方式投入抗原物质,即一次投入与分次投入,并观察了新产生滤泡数,维持的滤泡数,实验用小鼠２４ 只, 于足底注入铝和钥孔　血蓝素附合物（ＡＫＬＨ）, 分一次注入与三次注入组; 三次注入组又分间隔５ 日及间隔二周注入。注入后第３ 周与第１２ 周分别取出腘淋巴结,应用免疫组化法,第三周末观察可见一次投入产生的滤泡多, 而第十二周发现分次投入维持的滤泡数多。可见反应性滤泡的形成, 不仅与刺激物的性状和投入量有关, 而且与投入的方法有关     

IL-1 gene expression in lymphoid tissues

We examined the expression of IL-1 mRNA in vivo by in situ hybridization. RNA probes for murine IL-1 alpha and IL-1 beta were used to detect IL-1 mRNA in frozen sections of spleen, lymph node, and thymus of mice injected with Salmonella typhi LPS or SRBC. No IL-1 was detected in lymphoid tissues from un-injected mice. This lack of expression correlated with the absence of IL-1 biologic activity. However, after LPS injection, IL-1 alpha and beta mRNA expression was found in macrophages of the red pulp and marginal zone of the spleen. The periarteriolar lymphoid sheath contained cells that only expressed IL-1 beta mRNA. These cells were not lymphocytes and did not stain with the macrophage marker F4/80. A similar cellular response was found after SRBC injection. Scattered macrophages in lymph nodes and thymus were positive, but only after LPS or SRBC injection. The spleens of mice injected with LPS had megakaryocytes containing IL-1 mRNA.     

Ontogeny of the antigen-reactive lymph follicle-forming capacity of the popliteal lymph node in neonatal mice

The ontogenetic development of the reactive lymph follicle-forming capacity of the popliteal lymph node was investigated immunohistochemically in young mice which had received a single injection of hemocyanin (KLH) in a rear footpad at a predetermined age (between 1 and 21 days). The mice were sacrificed at various intervals after injection. In non-stimulated young mice, primary lymph follicles first appeared in the popliteal node at 11 days of age. When KLH was given to 7-day-old or older mice, each draining popliteal node showed a marked increase in B lymphocytes in the extrafollicular zone 3 days after injection and produced a number of "new" lymph follicles outside the pre-existing follicles over the next few days. In mice injected at 2-4 days of age, these nodes showed an increase in B lymphocytes in the outer cortex and had produced several lymph follicles by 8 days of age. The number of lymph follicles produced by each node tended to increase in line with age at injection. These results indicate that neonatal popliteal nodes become able to produce lymph follicles in response to exogenous antigens some time before ontogenetically developing follicles appear. The formation of new lymph follicles observed in draining popliteal nodes after KLH injection at an early postnatal age is discussed in relation to the ontogenetic development of stromal cells (precursors of follicular dendritic cells) that are capable of interacting with B lymphocytes and the extent of B lymphocyte influx into the node induced by KLH stimulation.     

Studies on immune responses to larval cestodes in mice: a simple mechanism of non-specific immunosuppression in Mesocestoides corti-infected mice

After intraperitoneal (i.p.) injection of sheep erythrocytes (SRBC) or dinitrophenylated Ficoll (DNP-Ficoll), mice infected with the larval cestode, Mesocestoides corti, contain at least 20x fewer antibody-secreting cells (PFC) in their spleens (or spleens plus lymph nodes) than uninfected mice. By contrast, intravenous injection of antigen leads to normal PFC responses. Results of studies on the fate of labelled syngeneic erythrocytes and foreign proteins suggest that i.p. injected materials are retained in the inflamed peritoneal cavity. Sequestration of antigen, and its subsequent local destruction, presumably accounts for the markedly suppressed systemic immune responses induced by i.p. injected antigens in M. corti-infected mice.     

The entry of T and B lymphocytes into rat popliteal lymph nodes undergoing a graft-versus-host reaction

The entry of radiolabeled blood-borne T and B lymphocytes into resting popliteal lymph nodes and popliteal lymph nodes stimulated with semiallogeneic lymphocytes was investigated in rats. Thoracic duct lymphocytes separated into T- and B-lymphocyte populations on nylon-wool columns were radiolabeled with 51chromium and equal numbers of T or B lymphocytes were injected intravenously. While the ratio of T and B lymphocytes in the blood is approximately 3:1 it was found that the ratio of T to B lymphocytes migrating into lymph nodes was approximately 9 T to 1 B lymphocyte in both resting and antigenically stimulated lymph nodes. Since the ratio of T to B lymphocytes in thoracic duct lymph is similar to that of blood, there is a disparity between the number of T cells entering and leaving lymph nodes. These results suggest that some T lymphocytes may return to the blood directly and/or there is increased T lymphocyte death in lymph nodes.     

Degree of chromatin activity and the RNA level in lymphocytes of popliteal lymph nodes of dogs after administration of an antigen]

In the experiment performed on 127 dogs by means of cytospectrofluorometric analysis, using fluorochrome acridine orange in dynamics up to 1 year, changes in the level of chromatin activation and RNA content have been studied in lymphocytes of the germinative centers and the crown of lymphoid nodules, in the paracortical zone and medullary cords of the regional and contralateral popliteal lymph nodes, after subcutaneous injection of antigen (BCG vaccine, 0.2 mg/kg) into the lateral area of the foot of the left pelvic extremity. The immune response is accompanied with a periodical increase in the level of chromatin activation and RNA content in populations of lymphocytes in the regional and contralateral popliteal lymph nodes with maximum in 6 h, 3-7 days, 1-3 months after the antigen injection. The intensity of these processes has an unequal level in lymphoid cells of various structural components; it is higher in lymphocytes of the contralateral lymph node.     

Secondary and Tertiary Responses of the Induced Bactericidin from the West Indian Spiny Lobster, Panulirus argus

West Indian spiny lobsters, Panulirus argus, synthesized a hemolymph bactericidin after being injected with killed suspensions of gram-negative bacillus EMB-1 isolated from the normal gut of this lobster. To study differences between the primary response and secondary response, animals were given a primary antigen injection of EMB-1 followed by a second injection of the same antigen 22 to 51 days later. As a rule, secondary bactericidal responses were enhanced over the primary in a manner reminiscent of specific anamnesis in mammalian immunoglobulin synthesis. Immunological memory was also suggested when tertiary responses were compared to secondary and by the persistence of residual titers for many days or weeks without additional antigenic stimulation.     

The extracorporeal immunostimulating effect of terrilytin in staphylococcal infection]

The intravenous injection of terrilytin-treated lymphocytes into rats infected with staphylococci enhances the formation of staphylococcal alpha antitoxin in the animals and the development of immune response to T-dependent antigen, such as sheep red blood cells (SRBC), but produces no effect on the development of immune response induced by T-independent antigen (lipopolysaccharide). Terrilytin-treated lymphocytes induce the release of the factor promoting the development of immune response to staphylococcal antigens and SRBC by spleen cells, incapable of adherence to plastic, but have no influence on the development of immune response to lipopolysaccharide in rats infected with staphylococci. At the same time in such rats spleen cells adhering to plastic take part in the transfer of signals from terrilytin-treated lymphocytes to nonadhering spleen cells of recipients.     

Kinetic immunochemical studies of IgG production during local and systemic anti-influenza immune response in rats

The kinetics of anti-influenza IgG antibodies in serum and nasal wash during the local and systemic immune response in rats was studied. The influenza virus A/HK/1/68 (H3N2) was injected by two different routes--intranasally and subcutaneously in the hind footpads. The proliferation of the Ig-forming cells in the popliteal and paratracheal lymph nodes either local or distant according to the mode of virus administration was also studied. The results obtained during the primary and secondary immune response suggested that an atypical immunization also produced a strong immune response in the distant lymph nodes. The nature of the secondary immune response supports the concept of migration of the activated lymphocytes from the peripheral lymph nodes to the natural portal of entry of virus thus giving rise to specific clonal population.     

Graft-versus-host reactivity of mouse thymocytes: effect of in vitro treatment with anti-TL serum.

The lymph ducts efferent from prefemoral nodes of sheep were cannulated and the lymph flow monitored during immune responses to injected allogeneic lymphocytes or xenogeneic murine P815 mastocytoma cells. Changes in the lymph began 5–6 days after injection of allogeneic cells but at 3–4 days after injection of xenogeneic cells, in both systems the number of large cells in the lymph increased to reach peak values of up to 40% of the total. The in vitro cytotoxic activity of lymph cells, cell supernatants, or cell free lymph was determined by measuring the release of ⁵¹Cr from prelabeled target lymphocytes or P815 cells. The cytotoxic mechanisms that were detected in the allogeneic and xenogeneic systems were similar; in both cases the lymph cells were cytotoxic only during the large cell response, and when the immunoblast numbers had returned to normal levels in the lymph no further cell-mediated cytotoxic effects were detected. During the blast response lymphocytes alone caused some target cell damage but their cytotoxic effector function was greatly increased in cultures containing complement or normal blood white cells. It was concluded that the lymph immunoblasts caused some target cell damage by direct action, but the majority of their cytotoxic activity was associated with synthesis and secretion of complement-dependent antibody (C.D.A.) and leukocyte-dependent antibody (L.D.A.).     

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.     

Enhancement of macrophage bactericidal capacity by antigenically stimulated immune lymphocytes

The ability of antigenically stimulated immune lymphocytes to influence the bactericidal capacity of normal macrophages was studied in vitro. Purified lymphocytes were obtained from the lymph nodes and peritoneal exudates of guinea pigs immunized with bovine gamma globulin (BGG) and from control animals. Immune and control lymphocytes were added to normal macrophages and incubated overnight in the presence or absence of BGG. After washing, the macrophage monolayers were infected with Listeria monocytogenes; 4 hr later, the cells were lysed and the surviving intracellular bacteria quantitated. The macrophages which had been incubated with BGG-immune lymphocytes in the presence of BGG displayed a markedly enhanced listericidal capacity. In parallel experiments, these same antigen-stimulated lymphocytes were shown to inhibit the migration of normal macrophages. Lymphocytes derived from peritoneal exudates were more active than lymph node lymphocytes in both assays.     

Humoral and cellular responses to native antigen following oral and parenteral immunization with lipid-conjugated bovine serum albumin

Humoral and cellular immune responses of rabbits to bovine serum albumin (BSA) were measured following oral and parenteral immunization with either BSA or one of two dodecanoic acid conjugates of BSA. The first consisted of a mixture of lightly and heavily conjugated BSA-molecules (L-BSA-mix), while the second (L-BSA) was a homogeneous preparation of heavily conjugated BSA with more than 95% of the 60 available amino groups covalently bound to dodecanoic acid. Animals ingesting L-BSA-mix had a similar humoral immune response but enhanced cellular reactivity to BSA in comparison to animals ingesting the native antigen. No systemic immunologic responses to BSA were detected following ingestion of L-BSA in spite of the demonstration of circulating BSA antigenic groups. This lack of a detectable immune response after oral administration was not due to masking of antigenic sites by the lipid residues since both humoral and cellular immune responses to BSA were obtained in animals injected with L-BSA. Ingestion of L-BSA did not induce tolerance since a subsequent injection of BSA elicited a normal primary immune response. The differences in immunogenicity between BSA, L-BSA and L-BSA-mix following oral administration may be related to different modes of antigen recognition by the gut-associated lymphoid tissues.     

Effect of antigen challenge on lymph node lymphocyte adhesion to vascular endothelial cells and the role of VLA-4 in the rat.

Lymphocytes from antigen-stimulated lymph nodes avidly migrate from the blood to cutaneous sites of inflammation such as DTH reactions or contact sensitivity. One of the initial steps in this migration is the adhesion of the lymphocyte to endothelial cells (EC); therefore, the adhesion of lymphocytes from antigen-stimulated lymph nodes to microvascular EC in the rat was examined. Two to five days after subcutaneous immunization with antigen, lymphocytes that adhered to unstimulated and IFN-gamma-, TNF-alpha-, IL-1 alpha-, and LPS-treated EC were increased in the regional lymph nodes. The enhanced adhesion was attributable to low-density lymphoblast-enriched lymph node cells while small high-density lymphocytes displayed little or no increase in their adhesion. Lymphoblast adhesion required the stimulation of the EC with 10 times the concentrations of IFN-gamma and TNF-alpha required for peritoneal exudate lymphocyte adhesion. There was a synergistic increase in the adhesion of the low-density lymphocytes to EC stimulated with combinations of IFN-gamma and TNF-alpha. Antibody to VLA-4 inhibited about 40% of the stimulated adhesion to EC treated with IFN-gamma, TNF-alpha, or LPS. In vivo anti-VLA-4 inhibited lymphoblast migration to IFN-gamma, TNF-alpha, LPS, and DTH reactions by 60%. Thus antigen stimulates the generation of low-density lymphoblasts that have an enhanced adherence to cytokine- and LPS-treated EC through a partially VLA-4-dependent mechanism and the migration of these cells to cutaneous inflammatory reactions is dependent upon VLA-4.