Induction and Maintenance of the Antibody Response by Different forms of Human Serum Albumin

Various forms of human serum albumin (HSA) were compared in their ability to induce and maintain the antibody response. In an in vitro model system, antibody synthesis was induced spontaneously during the maintenance phase of the immune response-presumably by persisting antigen. When different lymph nodes (LN) of the same rabbit were primed simultaneously with different forms of HSA, the spontaneous responses obtained in cell cultures prepared from LN primed with high molecular weight forms of HSA were greater than the responses obtained in cell cultures prepared from similar LN primed with lower molecular weight forms of HSA. This difference in response was consistent regardless of the method employed in antigen preparation and persisted for many months. The in vitro results indicating that the antibody response would be maintained at a higher level in animals immunized with high molecular weight forms of antigen and that the method of preparation would not be of major significance were confirmed in vivo in a mouse system.     

In Vitro Studies on the Effect of Anti-Lymphocyte Serum on the Humoral Immune Response

The effect of ALS (I), a heterologous anti-lymphocyte serum prepared against lymph node cells from rats pre-immunised with sheep erythrocytes (SRBC), on plaque forming cells (PFC) to SRBC was studied in vitro. ALS (I) reduced the number of both IgM and IgG PFC when complement was included in the reaction. This ability of ALS (I) to inhibit FFCs in vitro was absorbed out by the IgG fraction of anti-SRBC serum. Thus ALS (I) was thought to possess an anti-idiotypic antibody directed against B-cells at a later stage of differentiation.     

Antibody production in organ culture by bursae of mature chickens and the development of immunological competence

Antibody responsiveness of bursal lymphocytes was studied in vitro. Organ culture of bursal tissue in the presence of antigen, either sheep erythrocytes or bovine serum albumin, results in significant numbers of plaque-forming cells (PFC) compared to controls. The response in organ culture is age-dependent in that only bursae from chickens at least three weeks of age contained significantly increased numbers of secreting cells. Prolonged culture of normally unresponsive bursae from newly hatched birds results in a PFC response to antigen, suggesting that in vitro maturation occurs.     

Modulatory Effect of Isoprinosine on Lymphocyte Proliferative Response Under Immunodeppressive Conditions

In the present work the effect of Isoprinosine on the mitogenic responses of T and B lymphocytes has been studied. We have found that Isoprinosine can enhance in vitro the response to Concanavalin A. This enhancement was more apparent in cell cultures showing an initially low blastogenic response. In low responses artificially induced by treatments in vivo with cyclophosphamide, our results indicate that Isoprinosine, administered in vivo, does not enhance the response to Con A of treated mice. However, addition of Isoprinosine (75 μg/ml) to cultures of spleen cells from mice previously treated with cyclophosphamide enhanced the suppressed response up to normal levels. Neither in vivo nor in vitro Isoprinosine treatments increased the response of lymphocytes to lipopolysaccharide, but usually inhibited the blastogenesis of B cells.     

Immunological Properties of Peripheral Rat Lymphocyte Subpopulations Reacting Selectively with Guinea Pig Complement

Functional properties of peripheral rat T lymphocytes selectively affected by reaction with guinea pig serum (GPS) complement (C) were studied in this work. Cells sensitive to GPS cytotoxicity represented 2-7% of the nucleated cells in the spleen and 1-4% in lymph nodes. Responses of spleen and lymph node cells to Con A and PHA were markedly reduced following treatment with GPS whereas LPS-induced responses were not altered. GPS treatment also abrogated both the specific response of lymph node cells to a protein antigen and the production of leukocyte migration inhibitory factor by splenic cells. By contrast, GPS treatment significantly increased the reactivity of spleen and lymph node cells to alloantigens and enhanced the in vitro immune responsiveness of splenocytes to sheep erythrocytes. These results highlight the selective reactivity of guinea pig C with discrete subsets of immunoregulatory peripheral rat T lymphocytes.     

Localization of antibody-forming cells in draining lymphoid organs during long-term maintenance of the antibody response

Previous studies on the "spontaneous antibody response" have included in vitro steps and it is possible that the response is an in vitro artifact. The objective of the present study was to induce a spontaneous antibody response entirely in vivo and determine if the response is localized and if the magnitude of the response is related to the location of persisting antigen. Antigen was injected into the right hind footpads of mice, and lymph nodes on the right side were draining and lymph nodes on the left side were controls. Antibody-forming cells (AFCs) were enumerated in both draining and nondraining nodes 2 weeks, 2 months, and 1 year after secondary immunization. Four days prior to determining AFC number, the mice were severely bled to stimulate AFC production. Thousands of AFCs were found in the draining lymph nodes and the numbers were dramatic in nodes closest to the injection site that retain the most antigen. In contrast, the vast majority of nondraining nodes lacked any AFCs. One year after immunization, the response was almost exclusively in the popliteal node, draining the foot where antigen was administered a year earlier. These results are consistent with previous data on the spontaneous response and support the hypothesis that antigen retained on FDCs is essential in the maintenance of serum antibody levels.     

Induction of Differentiation of Human Null Cells into T Lymphocytes Under the Influence of Serum of Mice Treated with Sodium Diethyldithiocarbamate

Mouse serum collected 24 hr after administration of sodium diethyldithiocarbamate was shown to convert a subset of null cells, not bearing the human T lymphocyte differentiating antigen (HTLA^- cells) into HTLA⁺ cells in man. In addition to inducing surface chracteristics of T lymphocytes, the serum of treated mice increased fumtional activities of human peripheral blood lymphocytes, including the response to Con A and the suppressor activity in vitro.     

Induction and maintenance of the antibody response by different forms of human serum albumin.

Various forms of human serum albumin (HSA) were compared in their ability to induce and maintain the antibody response. In an in vitro model system, antibody synthesis was induced spontaneously during the maintenance phase of the immune response--presumably by persisting antigen. When different lymph nodes (LN) of the same rabbit were primed simultaneously with different forms of HSA, the spontaneous responses obtained in cell cultures prepared from LN primed with high molecular weight forms of HSA were greater than the responses obtained in cell cultures prepared from similar LN primed with lower molecular weight forms of HSA. This difference in response was consistent regardless of the method employed in antigen preparation and persisted for many months. The in vitro results indicating that the antibody response would be maintained at a higher level in animals immunized with high molecular weight forms of antigen and that the method of preparation would not be of major significance were confirmed in vivo in a mouse system.     

Mrphological and Functional Changes in Mouse Splenic Lymphocytes Following in Vivo and in Vitro Exposure to Chlorphentermine

With repeated administration to animals, the cationic, amphiphilic drug, cnlorphentermine (CP), has been shown by others to induce a phospholipidosis in lymphocytes. In the present study mouse splenic lymphocytes, exposed to CP, either in vivo or in vitro, developed morphological changes consistant With the induction of phospholipidosis. In addition, CP induced functional changes in lymphocytes. Mice, treated with CP in vivo, demonstrated a significantly depressed ability to generate a delayed hypersensitivity response or to produce antibody-secreting cells against de novo antigens. Mouse splenic lymphocytes, exposed to 10^-7 M CP for 3 days in vitro, demonstrated a signficantly depressed blastogenic response to the mitogens phytohemagglutinin, concanavalin A and lipopolysaccharide.

CP inhibited an event that occurred early during lymphocyte activation, but was subsequent to mitogen/receptor coupling. In addition, CP significantly depressed the increased uptake of choline that occurs in lymphocytes following cellular activation. Since the presence of phospholipidosis is indicative of an impairment in phospho lipid metabolism, these results taken together provide evidence for a relationship between this phenomenon and a ltered immune function.     

Induction and Maintenance of the Antibody Response by Different forms of Human Serum Albumin

Various forms of human serum albumin (HSA) were compared in their ability to induce and maintain the antibody response. In an in vitro model system, antibody synthesis was induced spontaneously during the maintenance phase of the immune response–presumably by persisting antigen. When different lymph nodes (LN) of the same rabbit were primed simultaneously with different forms of HSA, the spontaneous responses obtained in cell cultures prepared from LN primed with high molecular weight forms of HSA were greater than the responses obtained in cell cultures prepared from similar LN primed with lower molecular weight forms of HSA. This difference in response was consistent regardless of the method employed in antigen preparation and persisted for many months. The in vitro results indicating that the antibody response would be maintained at a higher level in animals immunized with high molecular weight forms of antigen and that the method of preparation would not be of major significance were confirmed in vivo in a mouse system.     

In Vitro and in Vivo Activity Profile of RHC 2851: A New Orally Effective Antiallergic Agent

RHC 2851 has been investigated for its antiallergic activity in three in vitro and two in vivo models of anaphylaxis. We have also compared its activity profile in these models with that of disodium cromoglycate (DSCG), doxantrazole, ketotifen and oxatomide. RHC 2851, given i.p., was 6 times more potent than DSCG, and given orally it was 3 times more potent than doxantrazole. As an inhibitor of mediator release, the activity profile of RHC 2851 was identical to that of DSCG in the following respects: inhibition of IgE-mediated in vitro release of histamine from rat mast cells (RMC) but not human basophils (HUB), possession of tachyphylactic properties and demonstration of rapid loss of inhibitory activity as a function of time before antigen challenge as well as inability to inhibit IgG₁-mediated release of histamine, both in vitro and in vivo, and lack of mediator antagonist activity. Ketotifen and oxatomide did not inhibit either IgE or IgG₁-mediated release of histamine in vitro or in vivo, and were potent mediator antagonists in vivo. We conclude that RHC 2851 is an orally effective inhibitor of mediator release with a mechanism of action similar to that of DSCG and different from that of ketotifen and oxatomide.     

Antibody isotypes mediating antigen retention in passively immunized mice.

Antibody isotypes vary in their capacity to mediate retention of a readily catabolized protein antigen, human serum albumin (HSA) in spleen, popliteal lymph node (PLN) and hind foot. Hyperimmune anti-HSA mouse sera were separated into fractions highly enriched for IgM, IgG1 and IgG2 via differential elution from protein A-Sepharose. These fractions were used to immunize normal mice passively. Twenty-four hours later the mice were injected with radio-iodinated HSA into the hind footpad. When the amount of HSA retained in the spleen 6 days later was determined, the potency of various antibody fractions to mediate retention could be ranked IgG2=IgG1 > IgM. The amount of HSA retention mediated by various fractions correlated well with autoradiographic evidence demonstrating localization of HSA in splenic follicles. The localization pattern in PLN was similar to the spleen except that the IgM-containing fraction mediated follicular localization of HSA to a considerable degree. In tendons of the hind foot, IgG1 mediated HSA retention five times better than IgG2 or IgM fractions. The amount of radioactivity found in the liver varied inversely with HSA retention in other locations. The results demonstrate differences in antibody isotype requirements for antigen localization in spleen, regional lymph node and collagenous sites of the hind foot.     

Antibody-dependent cellular cytotoxicity in poikilotherms

⁵¹Cr-chromate labelled chicken red blood cells, treated with rabbit (anti-chicken red blood cell) serum, are lysed in vitro, in the absence of complement, by spleen cells from Xenopus laevis, Ambystoma mexicanum or Lacerta viridis. Optimal conditions for lysis by Xenopus spleen cells were determined. The phenomenon seems homologous with antibody-dependent cellular cytotoxicity (ADCC) mediated by mammalian or avian K cells. The phylogenetic significance of the finding is discussed.     

Antigen-laden cells in thoracic duct lymph. Implications for adoptive transfer experiments

T cells from thoracic duct lymph of donor rats suppress the adoptive secondary response to human serum albumin (HSA). The original aim of the present investigation was to determine whether these non-immune cells have antigen-specific receptors. Thoracic duct lymphocytes (TDL) were depleted in vivo of antigen-specific T cells (negatively selected) by acutely injecting non-immune donors with HSA at the time of thoracic duct cannulation. Negatively selected TDL were mixed with memory cells (primed TDL from previously immunized donors) and transferred into irradiated recipients to assess whether the suppressive potential had disappeared. Paradoxically, the addition of negatively selected TDL (which were unresponsive to HSA) augmented the adoptive secondary anti-HSA response. Further study showed that the augmented response was mediated by a very small number of cells (~ 1 in 5000) laden with antigen that appeared in lymph of non-immune donors following HSA injection. These antigen-bearing cells were highly immunogenic and furthermore could overcome the effects of T suppressor cells in vivo. Once antigen laden cells were removed from lymph (by affinity chromatography), however, negatively selected TDL were found to inhibit the adoptive secondary response suggesting that either suppression in this model is non-specific or that antigen-specific suppressor cells are not selected out of the recirculating pool by antigen.     

Flow Cytometric Analysis of in Vivo Activation of Murine Spleen Cells

The in vivo effects of two polyclonal immune stimulators were studied in several strains of mice by analyzing the percentages of cells in various phases of the cell cycle by flow cytometry. Both lipopolysaccharide (LPS) and polyriboinosinic, cytidylic acid (rI.rC) were capable of inducing an increase in the percentage of cells undergoing DNA synthesis (S phase) in the spleens of several mouse strains. The response to both LPS and rI.rC was maximal between days 3 and 5 following injection. Optimal in vivo responses to LPS occurred at 3-30 μg, and to rI.rC at 100 μg; however, responses were observed over a broad dose range. No similar increase in S-phase cells was observed following injection of non-mitogenic T-independent antigens. Specific antibody was also measured after in vivo administration of rI.rC. There was a dissociation between the ability of an injection to induce specific antibody and to induce proliferation. These studies extend our knowledge of in vivo lymphocyte activation, and provide a basis for a detailed anafisis of lymphocyte activation following a variety of immune modulators in vivo.     

Immunosuppressive Characteristics of Human AFP: Effect on Tests of Cell Mediated Immunity and Induction of Humam Suppressor Cells

Murine AFP has been reported to be immunosuppressive in a variety of systems. However, the extent and degree of inhibition has varied in different species and laboratories. Therefore, we have examined the potential suppressive effect of purified human AFP on several in vitro tests of cellular immunity and the potential mechanism of its action AFP purified from fetal and liver cancer sera significantly inhibited mitogen and antigen-induced proliferative responses but had no effect on lymphocyte E rosetting, MIF production or mitogen induced T cell cyto-toxicity to Chang target cells. Purified human AFP induced human suppressor cell activity, capable of suppressing a one-way mixed lymphocyte reaction (MLC). In contrast to Con A induced suppressor cells, AFP induced suppressor cell activity was overcome by mitogen augmentation of the proliferative response in MLC. These data suggest that the inhibition of lymphocyte proliferation by human AFP may be mediated by the induction of a subpopulation of human suppressor cells. Furthermore, mitogen induced cell mediated cytotoxicity was partially inhibited by primary liver cancer serum and completely inhibited by newborn cord serum, in contrast to purified fetal or tumor AFP which had no effect. These data suggest that there are other immunosuppressive factors in fetal and tumor serum which require further characterization. These other serum factors may be responsible for some of the immunosuppressive effects attributed to AFP. Although AFP is unlikely to play a major immunosuppressive role physiologically in vivo, its selective effect on proliferative responses, apparently mediated by suppressor cells, may prove to be a useful pharmacologic probe of the mechanism of these in vitro lymphocyte responses and biological interactions.     

Modification of actin in peritoneal macrophages after diazepam treatment

We have investigated the effect of therapeutic doses of diazepam (7 μg/mouse) on the association of actin with the macrophage cytoskeleton using cytochemical and morphological methods.

Results obtained indicated that diazepam was able to modulate the content of actin in macrophages; such an effect proved to be time-dependent. After fixation and staining for indirect immunofluorescence with actin antibody, peritoneal macrophages from mice treated for short time with diazepam, showed a fluorescent intensity increase compared to control mice. The fluorescent intensity augmented reaching peak value within 14 days of treatment. Afterwards, this value dropped below control value for mice that underwent longer treatments. In the in vitro experiments concentrations of 10^-5 M, diazepam inhibited a well cell spread and a lower amount of actin after 15 min of incubation was also revealed.

These results suggest that administration of diazepam in vivo plays a role in both the nonspecific and specific immune response, producing in the macrophages a reorganization process of microfilaments.     

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.

     

Mycoplasma Neurolyticum Membranes: A T-Independent Antigen in the Rat

Our understanding of the mechanisms involved in B cell activation, proliferation and differentiation to immunoglobulin secreting cells has been facilitated by the use of T-independent and T-dependent antigens. The majority of these studies have used the murine system and only recently, the rat. Because membranes isolated from Mycoplasma neurolyticum are potent B cell mitogens in the rat and some T-independent antigens also activate DNA synthesis in B cells, the in vitro and in vivo antibody responses induced by M. neurolyticum membranes in T-deficient rat systems were examined. The three groups of rats used, i.e., nude; anti-thymocyte serum-treated, neonatally-thymectomized (ATS-Tx); and normal Fischer 344 produced a non-polyclonal antibody response against the membranes. Spleen cell cultures that were T cell deficient and B cell enriched produced plaque-forming cells against the Mycoplasma membranes. Antibody production was depleted upon removal of Sephadex G-10 adherent cells. The antibody response is comprised of both antigen-specific and polyclonal responses. Lipoglycan, found in the aqueous phenol extract of the membranes, is the mitogenic fraction of the membranes, and this study suggests that it may also be the T-independent antigenic component of the M. neurolyticum membranes.     

Restoration by Levamisole of Rosette-Forming Cells Inhibited by Histamine, DB-Camp and Adenosine

Db-CAMP and adenosine in vitro markedly inhibited the E-rosette formation of peripheral T-cells of healthy subjects. Histamine in vitro also inhibited the E-rosette formation, but only in patients with allergic disorders.

Levamisole in vitro significantly restored the E-rosette formation of T-cells inhibited by db-CAMP, adenosine and histamine.