Popliteal lymph node enlargement and antibody production in the mouse induced by zimeldine and related compounds with varying side chains

Structural requirements for induction of draining lymph node responses by the antidepressant drug zimeldine ((Z)-3-(4-bromophenyl)-N,N-dimethyl-3-(3-pyridyl)allylamine) in mice were determined by comparison of its activity with that of metabolites and analogues having different side chains. Mice received 1.0 mg of the compounds into the hind footpad and the popliteal lymph nodes (PLN) were removed 7 days later to determine weight, cell number and antibody production. Compounds with a methylated (Z)-(homo)allylamine side chain induced a marked PLN weight gain in C57BL/6 mice and a significant increase of the PLN cell number and of the IgM and IgG production per 10(6) PLN cells in BALB/c mice. Moderate PLN weight increase, but no significant antibody formation was induced by the doubly demethylated zimeldine metabolite, while compounds without an aliphatic amine or having a saturated side chain lacked significant activity in all assays. The (E)-diastereomer of zimeldine induced significantly less PLN weight gain than zimeldine in C57BL/6 mice, but an equal increase of PLN cell number in BALB/c mice. IgM and IgG responses to the (E)-diastereomer were moderate and absent, respectively. The antihistaminic drug, brompheniramine, having a saturated side chain and a 2-pyridyl ring, induced less PLN weight and cell gain than zimeldine and failed to increase antibody formation. The capacity of the compounds to induce PLN responses appeared not related to their pharmacological potential to inhibit the reuptake of serotonin and noradrenaline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of popliteal lymph node enlargement and antibody production in the mouse by pyridylallylamines related to zimeldine

The role of the para-brominated phenyl ring of the allylamine antidepressant drug, zimeldine, in its immunomodulating activity was studied by comparing local lymph node responses of mice to analogues with various phenyl ring substituents. The compounds (1.0 mg) were injected into the hind footpad and weight and cell number of the popliteal lymph node (PLN) and antibody production by PLN cells were determined 7 days later. Zimeldine and the para-trimethylsilylated and ortho-para-chlorinated compounds induced a marked PLN weight gain in C57BL/6 mice. The para-chlorinated and meta-brominated analogues were significantly less effective, while the para-fluorinated and the unsubstituted analogue failed to trigger PLN weight gain. The same range of potency of the compounds was seen when increases of PLN cell number in BALB/c mice and increases of IgM production by a fixed number of BALB/c PLN cells were determined. The IgG production on a per cell basis was not increased by the fluorinated and the unsubstituted compounds, while the remaining compounds stimulated the IgG production to a similar extent. Plots of the lipophilicity of the para-substituted compounds against their ability to induce PLN weight and cell gain showed a fair correlation, but induction of antibody formation tended to be counteracted at too high lipophilicity. No correlation between pharmacologic and immunologic activity of the compounds could be found. Data indicate that lipophilicity grossly favours PLN enlargement, but the divergent responses to the equilipophilic 3- and 4-brominated compounds, suggest conformational restraints as well.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-term antigen retention by dendritic cells in the popliteal lymph node of immunized mice.

Antigen retention by follicular dendritic cells (FDC) was studied in the popliteal lymph nodes (PLN) of mice actively or passively immunized against human serum albumin (HSA) or horse spleen ferritin. Electron microscopic autoradiography was used to locate a challenge dose (1 microgram) of 125I-labelled HSA in the draining PLN following injection into the hind footpad of specifically immune mice. In both actively and passively immunized mice, the radiolabelled antigen was localized to the follicles in the cortex of the draining node. In actively immunized mice, the antigen formed a crescent of label on the superficial aspect of germinal centres, while in passively immunized mice label was seen in primary follicles. In electron microscope autoradiographs, the silver grains were concentrated in areas of dendritic cell processes which emanated from a cell body containing a characteristic irregular nucleus. The size and complexity of the dendritic cell processes increased in actively immunized mice suggesting that the FDC could hypertrophy. High resolution studies using the electron-dense antigen, ferritin, showed that it was localized to the extracellular space of the FDC processes and was associated with amorphous electron-dense material; presumably immune complexes. Antigen was not present uniformly distributed in the extracellular material but rather it was in discrete patches occupying small segments of the FDC processes. Large amounts of label-free electron-dense material were present suggesting that immune complexes of various specificities were present on each DC. Ferritin was seen more than 3 months after challenge but only on the FDC. The data suggest that the antigen retaining mechanism in the lymph node of immune mice is antigen non-specific, is capable of hypertrophy in response to active immunization and provides a mechanism for stable long-term retention of antigen.     

Antigen in tissues: IV. The effect of antibody on the retention and localization of antigen in rat lymph nodes*

A study has been made of the role of specific antibody in causing increased retention and specific localization of a weak antigen, human serum albumin (HSA), in the popliteal and aortic lymph nodes of rats. The antigen was labelled with ¹²⁵I prior to mixing with antibody. HSA mixed with excess homologous antibody was trapped to the greatest extent in these nodes after footpad injection of the antigen. Injection of HSA with antibody caused increased uptake of HSA into the medulla but retention was poor as autoradiographs showed the area to be essentially free of antigen 4–5 days after injection. By contrast, antigen injected with antibody localized strongly in lymphoid follicles and persisted at this site. Both IgM and IgG antibody were found to cause follicular localization of HSA in rats. Heterologous, isologous and homologous antibody also caused follicular localization of the antigen. Purified homologous γ-globulin was found to localize in the follicles. A moderate increase in the net negative charge of the γ-globulin by acetylation did not appreciably affect the ability of the globulin to localize in the follicles. Detectable formation of antibody did not occur in the rats after injection of antigen—antibody complexes, owing possibly to the inhibitory effect of free antibody on the primary response.     

A Murine Model for Analysis of Spontaneous Induction and Feedback Regulation of Specific Antibody Synthesis

A mouse model system has been developed in which antibody specific for human serum albumin (HSA) was produced spontaneously in irradiated recipients of lymph node cells. This response occurred in the absence of exogenous HSA when draining lymph node cells from mice immunized months earlier were adoptively transferred. This spontaneous response was inhibited by administration of rabbit anti-HSA indicating that rabbit antibody can mediate feedback suppression in the mouse. Transfer of non-draining lymph node cells or spleen cells, even from the same mice, resulted in insignificant spontaneous induction of anti-HSA. The antibody levels in recipients of draining lymph node cells increased with time up to 15 days after adoptive transfer and were directly proportional to the number of cells transferred. Incubation of donor cells in vitro was not required for spontaneous induction but generally enhanced spontaneous responses in recipients. Secondary immunization of the donor mice also enhanced the response. We attribute this spontaneous induction of antibody synthesis to antigen persisting in the draining lymph nodes. It appears that persisting antigen may play a major role in the maintenance and regulation of serum antibody levels. This mouse system is amenable to both in vitro and in vivo manipulations. This flexibility makes it an attractive model for studying the role of persisting antigen and other factors responsible for the maintenance and regulation of serum antibody.     

Immune elimination and immune retention: the relationship between antigen retained in the foot and the elicitation of footpad swelling.

We determined whether (1) long term antigen retention is present in distal sites after footpad challenge of immune mice; (2) if antigen retained in the foot is selectively localized; and (3) if the foot is adversely affected by the retained antigen. Mice immune or non-immune to human serum albumin (HSA) were injected in the hind footpads with 125I-HSA. In immune mice rapid clearance of radiolabel occurred in the liver, lungs, kidney, blood and urine but radiolabel was retained in the hind feet, draining lymph nodes and spleen. Non-immune mice rapidly cleared radiolabel from these sites. Autoradiography revealed that most of the radiolabel in the feet was in flexor tendons and tendon sheaths. Electron microscope autoradiography indicated that antigen was associated with collagen at the tendon surface, but not with cells or cell processes. Radiolabel solubilized from the feet, lymph nodes and spleen could be specifically precipitated with rabbit anti-HSA. Histological examination of the tendon and surrounding tissues did not show that retained antigen was causing inflammation or chronic tissue damage. Nanogram levels of antigen could elicit swelling in the sensitized foot even if the antigen was injected at a remote site, but mice immunized by other routes or against other antigens did not show footpad swelling. Antigen retained on collagenous tissues may induce hypersensitivity and thus play a role in rheumatic diseases.     

Popliteal lymph node enlargement and antibody production in the mouse induced by drugs affecting monoamine levels in the brain

Drugs that affect monoamine levels in the brain were screened for their potential to cause immunological changes in the popliteal lymph node (PLN) of mice after injection into the hind paw. The tricyclic antidepressant (TCA) drugs, imipramine and amitriptyline, and the serotonin reuptake blocker, zimeldine, induced a prominent PLN weight gain in C57BL/6 mice. Ketanserin and ritanserin appeared less effective while nomifensine, serotonin and the antigen, sheep erythrocytes, lacked significant activity. In BALB/c mice all agents induced an increase of PLN cell number, the TCA drugs and zimeldine appeared superior in this respect. Increased IgM as well as IgG production on a per cell basis was only induced by the TCA drugs, zimeldine and, especially, sheep erythrocytes. Data indicate that induction of PLN responses is not a general property of agents affecting monoamine levels. Structural, i.e. antigenic, characteristics of the drugs rather than their pharmacological properties are probably at play.     

A potential weak allelic difference in male-specific antigen between two inbred strains of mice

Differences were detected between male-specific antigen(s) from BALB/c and C57BL/10 mice. Reciprocal crosses between these strains were analysed by means of a popliteal lymph node assay enumerating cells secreting IgM and IgG in a T-dependent bystander B-cell response. The responses, while being indirect, weak and variable, suggested that there are differences in 'immunogenicity' coded by Y-linked (male-specific) genes. This conclusion was strengthened by the results of experiments carried out in Y-backcross mice, where spleen cells from male C57BL mice with a BALB/c-Y (B10.C-YC) stimulated low popliteal lymph node responses in female littermates in comparison with C57BL mice with their own Y-chromosome. In contradistinction, male spleen cells from a BALB/c with a C57B1/10-Y chromosome (C.B10-YB), injected into a hind footpad of a female littermate, induced a relatively higher popliteal lymph node response.     

Elimination of spleen and of lymph node macrophages and its difference in the effect on the immune response to particulate antigens

To study the role of macrophages in the in situ immune response to particulate antigens in spleen and popliteal lymph nodes (PLN), mice were injected with dichloromethylene diphosphonate (Cl2MDP)-containing liposomes to eliminate macrophages, followed by immunization with trinitrophenylated sheep red blood cells (TNP-SRBC). Depletion of macrophages in the spleen caused a strong decrease in the number of antibody-forming cells (AFC), which develop after intravenous (i.v.) injection of the antigen. These results strongly suggested the involvement of splenic macrophages in the processing of TNP-SRBC. In particular, the populations of marginal zone macrophages may be involved in the inductive phase of an antibody response to particulate antigens. These macrophages are strategically positioned at the end of the white pulp capillaries in the marginal zone of the spleen and they have their cell processes between the marginal zone-B cells. Elimination of macrophages in PLN had no effect on the number of AFC, which develop after subcutaneous (s.c.) injection of the antigen in the hind footpads. This indicates that the macrophages are not essential for the induction of a local immune response to the particulate antigen TNP-SRBC. After depletion of lymph node macrophages, the number of AFC developing in the spleen after s.c. footpad injection of the antigen increased and the anti-TNP serum titers were elevated. This may well be caused by the fact that more of the antigen reaches the circulation and subsequently stimulates the spleen.     

Murine lung immunity to a soluble antigen

Although it is known that soluble antigen is immunogenic when deposited in the respiratory tract, less is known about lung immunity to soluble antigen than is known about lung immunity to particulate antigen. To test the hypothesis that soluble antigen triggers antigen-specific immunity in the respiratory tract in a fashion similar to that reported for particulate antigen, we examined the development of local and systemic immunity in C57BL/6 mice after intratracheal (i.t.) instillation of a soluble, large molecular weight protein neoantigen, keyhole limpet hemocyanin (KLH). Specific anti-KLH IgG and IgM first appeared in the sera of mice on day 7 after primary immunization by i.t. instillation of KLH, with specific serum antibody concentrations remaining elevated at day 11. Cell populations prepared from lung-associated lymph nodes of immunized mice released specific anti-KLH IgG and IgM in vitro; peak levels were obtained from cells isolated 7 days after antigen instillation, with levels of specific antibody released by cells isolated on days 9 and 11 decreasing markedly. Cultured spleen cells obtained from mice after primary immunization released only low levels of specific IgM, and no specific IgG. No specific antibody was released by cell populations derived from the lungs of animals undergoing primary immunization. When presensitized mice were given an i.t. challenge with KLH, responses differed markedly from those following primary immunization. Lung-associated lymph node cell populations from challenged mice released greater amounts of specific antibody earlier than did cell populations from mice undergoing primary immunization.(ABSTRACT TRUNCATED AT 250 WORDS)     

In Vitro Regulation of the Anamnestic Antibody Response Upon Addition of Heterologous Antigens to Primed Lymphoid Cells

Alum-precipitated keyhole limpet hemocyanin (KLH) and polymerized human serum albumin (HSA) were injected into the hind foot pads of rabbits. Many months later the popliteal lymph nodes were removed and cell cultures prepared and incubated in media containing different concentrations of KLH or native HSA. These primed cells responded to some concentrations of KLH with the synthesis not only of homologous antibody but of heterologous IgG antibody to HSA. They also responded to some concentrations of HSA with the production of heterologous antibody to KLH as well as homologous antibody. In some experiments the addition of KLH induced the cells to synthesize as much antibody to HSA as did the addition of HSA itself. The nonspecific stimulation of the anamnestic antibody response to HSA required the cultures to contain KLH, HSA, KLH-reactive memory cells and, presumably memory cells reactive with HSA.

The suspension of KLH-and HSA-primed cells responded to other concentrations of KLH or egg albumin (EA) with inhibition of the antibody response to HSA. This non-specific inhibition did not require lymph node cells which had been primed by the previous injection of KLH or EA.

KLH and HSA did not cross-react with respect to immunogenicity in vitro or in vivo or reactivity with antibody in vitro.

According to a model which was proposed, the KLH induced KLH-reactive T lymphocytes to produce soluble factor(s) which regulated the response of HSA-reactive B lymphocytes to HSA-specific determinants which persisted from the original injection.     

In Vitro Regulation of the Anamnestic Antibody Response Upon Addition of Heterologous Antigens to Primed Lymphoid Cells

Alum-precipitated keyhole limpet hemocyanin (KLH) and polymerized human serum albumin (HSA) were injected into the hind foot pads of rabbits. Many months later the popliteal lymph nodes were removed and cell cultures prepared and incubated in media containing different concentrations of KLH or native HSA. These primed cells responded to some concentrations of KLH with the synthesis not only of homologous antibody but of heterologous IgG antibody to HSA. They also responded to some concentrations of HSA with the production of heterologous antibody to KLH as well as homologous antibody. In some experiments the addition of KLH induced the cells to synthesize as much antibody to HSA as did the addition of HSA itself. The nonspecific stimulation of the anamnestic antibody response to HSA required the cultures to contain KLH, HSA, KLH-reactive memory cells and, presumably memory cells reactive with HSA.

The suspension of KLH-and HSA-primed cells responded to other concentrations of KLH or egg albumin (EA) with inhibition of the antibody response to HSA. This non-specific inhibition did not require lymph node cells which had been primed by the previous injection of KLH or EA.

KLH and HSA did not cross-react with respect to immunogenicity in vitro or in vivo or reactivity with antibody in vitro.

According to a model which was proposed, the KLH induced KLH-reactive T lymphocytes to produce soluble factor(s) which regulated the response of HSA-reactive B lymphocytes to HSA-specific determinants which persisted from the original injection.     

Antigen in tissues: V. Effect of endotoxin on the fate of, and on the immune response to, serum albumin and to albumin—antibody complexes*

Bacterial endotoxin was injected into rat hind footpads together with bacterial flagellin and ¹²⁵I-labelled human serum albumin (HSA); the latter was used unmodified or heat denatured (H.HSA) or as an HSA—antibody complex. Endotoxin did not affect the trapping, retention nor localization of the labelled HSA in the popliteal and aortic lymph nodes, whether the antigen had been injected as HSA, H.HSA or as an HSA—antibody complex.

If endotoxin was injected at the same time as: (1) flagellin, there was an increased production of anti-flagellin antibody; and (2) H.HSA or HSA—antibody complex, detectable amounts of anti-HSA antibody were produced. When H.HSA and endotoxin were injected, the primary response was long lived yet the period of induction of antibody formation and of antigen persistence in the lymphoid tissues was short. If, during the primary antibody response to H.HSA, the animals were challenged with HSA, equally strong secondary antibody responses occurred with an HSA—antibody complex or with HSA alone.

The results were interpreted in terms of the tissue localization pattern of H.HSA (medullary macrophage) and HSA—antibody complex (medulla and lymphoid follicles). It was suggested that: (1) induction of antibody formation and priming of cells for a secondary antibody response might occur following localization of the antigen in the medulla and that antigen localization in the lymphoid follicles might not be a strict requirement for this; and (2) the follicular localization of antigen might be the preferential mechanism for the firing of a secondary antibody response.

     

The induction and migration of antigen-specific helper cells for IgA responses in the intestine.

The distribution of keyhole limpet haemocyanin (KLH)-specific helper cells for antibody responses of IgA, IgM and IgG isotypes in Peyer's patch (PP), mesenteric lymph node (MLN) and peripheral lymph node (PLN) was examined following oral, intraduodenal (ID), intraperitoneal (IP), intra-Peyer's patch (IPP) or subcutaneous (SC) immunization with KLH. Oral or ID immunization gave little or no response in any tissue studied. IP immunization with or without a subsequent ID challenge gave rise to a modest IgA and IgM helper response in MLN but a small IgA and IgM helper response in PP and PLN. IP immunization alone did not stimulate IgG-specific help in any tissues studied, but a small IgG helper response occurred in MLN and PLN after subsequent ID challenge. IPP was the most effective route of immunization, giving rise to a large helper response for IgA, IgM and IgG isotypes in PP, a smaller response in MLN and no response in PLN. The helper response following IPP immunization was not augmented by subsequent ID challenge. SC immunization gave a small but significant helper response for all isotypes in PLN but no response in PP or MLN. The kinetics of the helper response were examined in PP, MLN, PLN and thoracic duct lymph (TDL) following IPP immunization. The helper response for all isotypes in PP was maximal at 2 weeks and then declined. Similar kinetics but of lower magnitude were observed in MLN and TDL. The presence of IgA-specific helper cells in TDL demonstrates that these cells migrate, presumably from GALT, and may constitute an important component of mucosal responses at extraintestinal sites.     

Depression of the immune response to sheep erythrocytes in mice infected with Taenia crassiceps larvae.

Intraperitoneal infection of mice with larvae of the cestode parasite Taenia crassiceps results in depression of both primary and secondary antibody responses to sheep erythrocytes in vivo. The depression is not associated with a shift in kinetics of the response. Both immunoglobulin M (IgM) and IgG responses are depressed, but IgG is depressed more than IgM in the secondary response. Secondary in vitro sheep erythrocyte responses are consistently depressed in both spleen and mesenteric lymph node cell preparations from infected mice, whereas primary in vitro sheep erythrocyte responses are consistently depressed in mesenteric lymph node cell preparations but not always in spleen cell preparations. These results are consistent with antigenic competition. The cell type or types involved in mediation of the immunological defect in the infected animals remain to be identified.     

Lymphocytes from enlarged iliac lymph nodes as fusion partners for the production of monoclonal antibodies after a single tail base immunization attempt

A novel method of preparing hybridomas producing mouse monoclonal antibodies was -established, called "the mouse iliac lymph node method". Lymphocytes from enlarged iliac lymph nodes from mice injected intramuscularly at the tail base with an emulsion of antigen and Freund's adjuvant were used for cell fusion. For the most part, lymph node lymphocytes from two mice were used for a single cell fusion attempt. Ovalbumin was used as the antigen and the results of fusion were compared with the results of a previous report (Cell Struct. Funct. 20; 151-156, 1995). Approximately 100 positive wells producing antibody of interest were identified using this method. By comparison, approximately 10 positive wells were identified using the more conventional mouse spleen method after three immunization injections. The relative proportions of hybridomas producing IgM, IgG1, IgG2a, IgG2b, and IgG3, following fusion using iliac lymph node lymphocytes obtained 14 days after injection were 14.0%, 78.7%, 3.2%, 3.5% and 0.5%, respectively. This method demonstrated the following advantages: (1) a single injection of the antigen emulsion was sufficient, (2) the lymph nodes were ready for use 14 days after injection, and (3) a high yield of positive hybridomas was obtained.     

Cells involved in the immune response. XXXI. The role of the spleen in the primary and secondary immune responses in the normal adult outbred rabbit: the initial localization of memory cells to the spleen and their subsequent dissemination to the thymus and peripheral lymph nodes

Normal adult outbred rabbits were immunized intravenously (iv) with sheep erythrocytes (SRBC). At varying times thereafter, the different lymphoid organs were investigated for spontaneous and culture-induced antibody secreting cells by the aqueous hemolytic plaque-forming cell (PFC) technique. During the phase of active antibody formation (Days 3 to 30), immediate PFC, indicative of spontaneous antibody synthesis and secretion, were detected principally in the spleen. In the early postimmune memory period (Days 30 to 90), memory cells capable of generating PFC following secondary immunization in in vitro culture with SRBC were detected only in the spleen. However, by 4 months postimmunization, memory cells were detected in the thymus and popliteal lymph node (PLN) as well as in the spleen. The number of memory cells in the thymus and PLN was significantly higher by 6 months postprimary iv immunization and was even further elevated by 9 months postprimary iv immunization. Following in vivo secondary immunization by the iv injection of SRBC 2 or 6 months postprimary immunization, immediate PFC were detected in large numbers in the spleen, the bone marrow, and the blood, marginally in the PLN and not at all in the thymus. Similar results were obtained at 9 months following primary immunization with SRBC with the exception that large numbers of immediate PFC were detected in the PLN following secondary iv immunization. Following culture of these lymphoid cells for 5 days in vitro with SRBC, the thymus and PLN cells, as well as the spleen cells, generated large numbers of PFC. Since immediate PFC were never detected among the freshly isolated thymus cells whereas thymic cell cultures 6 and 9 months postprimary iv immunization invariably generated large numbers of PFC following secondary immunization in vitro, the thymus memory cells would appear to be inaccessible to particulate antigen injected intravenously; they can only be detected following activation by the antigen in culture. The PFC generated by thymus memory cells (and spleen and PLN) were totally inhibited by the inclusion of sheep anti-rabbit IgG into the PFC assay. This finding demonstrates unequivocally that the plaques induced by thymus cells, just as the plaques induced by spleen and PLN cells, are antibody mediated and not false plaques. Therefore, the thymic PFC cells must be antibody-secreting B-memory cells since T cells do not synthesize or secrete immunoglobulins.     

Retention of Immune Complexes by Murine Lymph Node or Spleen Follicular Dendritic Cells

Using monoclonal anti-trinitrophenyl (TNP) antibodies complexed to TNP-myoglobin-coated gold particles, we analysed at the ultrastructural level the retention by follicular dendritic cells (FDC) of immune complexes containing various antibody isotypes. Gold-labelled immune complexes were injected subcutaneously or intravenously into naive mice and, after 24 h, germinal centres of draining lymph nodes or spleen were examined by electron microscopy. FDC generally retained complexes containing IgG2a and IgG2b better than those formed with IgG1 or IgG3. IgM was rarely retained. FDC isolated from lymph nodes or spleens were incubated in vitro with gold-labelled complexes in a serum-free medium. IgG2a and IgG2b complexes were also retained in vitro in large quantities by FDC; IgG1 and IgG3 complexes were retained in smaller quantities or in highly variable quantities compared with IgG2; IgM complexes were rarely seen on FDC. There was no difference between FDC isolated from lymph nodes or from spleen with respect to the Ig isotypes required for Fc-mediated retention of immune complexes.     

Prolonged antigen half-life in the lymphoid follicles of specifically immunized mice

The kinetics of clearance of ¹²⁵I from the popliteal lymph nodes and feet of human serum albumin (HSA)-immunized mice was studied following the injection of [¹²⁵I]-HSA into the hind footpads. Antigen was cleared from both locations rapidly for the first few days. The antigen half-life (T½) during this period was only a matter of hours. By the end of the first week, however, the rate of clearance in both sites had changed markedly. The antigen T½ in the node between the first and sixth week was 8.1 weeks (95% confidence interval between 5.1 and 20 weeks) and the antigen T½ in the foot was 6.1 weeks (95% confidence interval between 3.7 and 16.6 weeks). There was, however, about twenty times more radioactivity in the feet than in the popliteal nodes. Autoradiography of popliteal lymph nodes revealed that initially antigen was trapped in the medulla, subcapsular sinus, superficial cortex and around lymphoid follicles. During the first few days antigen was cleared from all sites except the follicles. The radioactivity initially trapped in the medulla, subcapsular sinus, and superficial cortex appeared to have been associated with macrophages. Studies with peritoneal macrophages indicated an antigen T½ in these cells of 2 h (95% confidence interval between 1.5 and 3 h). The initial rapid clearance of antigen trapped and catabolized by macrophages and the long-term retention of antigen in the follicles is probably attributable to trapping and retention by follicular dendritic cells. The large pool of antigen trapped in the foot did not appear to serve as a depot to replace antigen degraded in the node, since amputation of the foot did not alter the level of antigen retained in the node. The long antigen T½ in the lymph node follicles indicates that antigen is available in the lymph node to play a role in the maintenance and regulation of immune responses for many months or even years.     

Use of Peyer''s patch and lymph node fragment cultures to compare local immune responses to Morganella morganii.

Lymphoid tissue fragment cultures were established to analyze the differentiative processes among B cells in Peyer's patches (PP) and peripheral lymph nodes (PLN), especially those in germinal centers. PP cultures from both conventionally reared mice and formerly germ-free mice colonized with Morganella morganii could be maintained for greater than 12 days with continued B-cell division, especially among cells binding high levels of peanut agglutinin, a characteristic of germinal center cells. PLN cultures from conventionally reared mice injected with a heat-killed vaccine of M. morganii could be maintained for the same amount of time. Over this period, PP cultures continued to secrete immunoglobulin A (IgA) as well as smaller amounts of IgM. PP cultures from formerly germ-free mice colonized with M. morganii showed net increases of IgA antiphosphocholine (anti-PC) antibodies with avidities as high as those of the prototypic T15 monoclonal antibody. Similar PLN fragment cultures from conventionally reared mice given footpad injections of M. morganii showed net increases of IgM and IgG anti-PC antibodies in the culture fluid. Thus, although M. morganii stimulated lymphoid tissues in vivo to produce an anti-PC response in vitro when given by either the oral or the parenteral route, the antibody isotypes differed between PP and PLN fragment cultures. Fragment culturing may offer a complementary and simpler way to detect a local secretory IgA response than does either measuring IgA antibody in secretions or detecting IgA antibody in the cytoplasm of plasma cells in the lamina propria of gastrointestinal or respiratory tissue.