IgG1 antibody-dependent mediator release after passive systemic sensitization of basophils arriving at cutaneous basophil hypersensitivity reactions

When antigen is injected into a 24-hr cutaneous basophil hypersensitivity (CBH) reaction of an actively sensitized guinea pig, local basophils degranulate and release histamine. This reaction is called cutaneous basophil anaphylaxis and may be antibody mediated. We now report passive sensitization of basophils at CBH sites by systemic transfer of anti-picryl immune serum. Keyhole limpet hemocyanin- (KLH) immunized animals were skin tested with KLH to elicit 24-hr CBH reactions at day 7. Anti-picryl serum was injected i.v. at various times. On day 7, blue dye was injected i.v., and then 24-hr CBH sites vs nearby normal skin were challenged with 0.1 microgram picryl-human serum albumin (Pic-HSA). An immediate increase in vascular permeability (blueing) was noted at normal skin sites due to systemic passive cutaneous anaphylaxis (PCA), and augmented blueing occurred at CBH sites compared with normal skin. Systemic passive sensitization of CBH sites occurred when antiserum was administered as little as 1 hr before challenge of CBH site. However, local administration of anti-picryl serum (as in a local PCA reaction) was not able to sensitize tissue basophils, whether antigen was administered locally or systemically. The serum factor that mediated cutaneous basophil anaphylaxis was heat-stable (56 degrees C X 4 hr) 7S IgG1 antibody. Electron microscopy of Pic-HSA-challenged CBH sites in animals that received IgG1 antibody showed that local basophils undergo anaphylactic degranulation by exocytosis. These studies suggest that basophils arriving at CBH reactions are sensitized for anaphylactic function by antibody that can be acquired in the circulation, but possibly not at the local site.     

The effect of synthetic protein fragments on humoral immune response

Effect of some protein and immunopeptide synthetic fragments on the humoral immune response has been studied. Some investigated peptides are shown to influence the antibody genesis at the secondary immune response to T-dependent antigen (bovine serum albumin) in mice. The serum antibody level specific for the bovine serum albumin is measured by enzyme-linked immunosorbent assays (ELISA). Possible mechanisms of the influence of such peptide fragments on the immune system is discussed.     

A newly described activity of guinea pig IgG1 antibodies: transfer of cutaneous basophil reactions.

Hapten-specific delayed time course skin reactions containing predominant accumulations of basophils and eosinophils were elicited in newborn guinea pigs after i.v. transfer of small amounts of oxazolone immune serum. The immune serum was fractionated by column chromatography procedures, and the fractions were examined for their ability in transferring this form of cutaneous basophil hypersensitivity (CBH). Only the 7S IgG-containing peak from Sephadex G-200 columns, and only the IgG1-containing fractions from DEAE columns, transferred CBH. An affinity column of bound oxazolone removed the activity from immune serum, and it could be recovered from the column by eluting with soluble oxazolone. About 35 microgram of purified IgG1 anti-oxazolone antibody could systemically transfer CBH reactivity. An immunoadsorbant column of anti-IgG1 removed this activity, but a column of anti-IgG2 did not. None of the procedures were able to separate activity in transferring CBH from passive cutaneous anaphylactic (PCA) activity classically associated with guinea pig IgG1 antibody. IgG1 from 8-day immune and 31-day hyperimmune donors were both effective. The average association constant of 8-day antibody was 8 X 10(-4) M-1. Transfer of cutaneous basophil reactions can be mediated by low affinity serum 7S IgG1 antibody.     

Systemic expression of cutaneous basophil hypersensitivity.

Guinea pigs primed for cutaneous basophil hypersensitivity (CBH) with several soluble proteins or with sheep erythrocytes developed a systemic, delayed-onset, maculopapular rash when challenged parenterally with specific antigen. The rash was most readily induced 5 to 7 days after immunization, at a time when local CBH skin test reactivity was also optimal. Miscroscopically, the rash resembled local CBH skin test reactions, being comprised of a papillary dermal infiltrate of basophils and lymphocytes and a striking dilatation and compaction of superficial venules. In addition to the systemic rash, animals expressing systemic CBH (SCBH) exhibited a striking eosinophilia at 24 hr which gave way to basophilia at 48 hr. Focal collections of eosinophils, and of smaller numbers of basophils, were found in the lungs and spleen; both eosinophils and basophils infiltrated the medulla of the thymus. Thus, basophil-rich infiltrations are favored in the skin even after systemic challenge with antigen and occur only to a much smaller extent in other organs where eosinophils may predominate. These differences in the response of various organs to challenge with parenteral antigen suggest that as yet unidentified local factors play a determinative role in regulating the inflammatory response. The pathogenesis of SCBH is not yet established, but it shares many of the properties of local CBH: histology, carrier specificity, development early after sensitization in the absence of detectable antibodies. Passive transfer has not been accomplished with serum alone but has been achieved irregularly with cells plus serum. SCBH may serve as a useful model for several disease states in man characterized by a systemic rash and eosinophilia, including certain types of drug reaction.     

In vitro evidence indicating a role for the Fc region of IgG in the mechanism for the long-term maintenance and regulation of antibody levels in vivo

The synthesis of anti-human serum albumin (HSA) antibody was induced spontaneously in cell cultures prepared from the draining lymph nodes of rabbits immunized months earlier with polymerized HSA. Serum from HSA-immunized rabbits suppressed this response. Removal of specific antibody from immune serum eliminated suppression and the addition of specific IgG restored suppression, indicating that the feedback phenomenon may be explained by an effect of specific IgG antibody. Fab and F(ab′)₂ fragments masked antigen as effectively as IgG; however, they were markedly inferior to IgG in mediating suppression. Furthermore, Fab competed with IgG and interfered with IgG mediated suppression. The addition of small amounts of antigen to antibody-suppressed cultures induced an antibody response. The level of induction was proportional to the antigen-antibody ratio. However, 80 to 100 times the antibody concentration needed to mask all antigenic determinants was needed in order to eliminate induction of antibody synthesis. High concentrations of antigen-antibody complexes at equivalence also suppressed the spontaneous response. This suppression was similar to antibody mediated suppression at the spontaneous response in that the Fc region of IgG was required.     

Response and function of cutaneous mucosal and serum antibodies in barramundi (Lates calcarifer) acclimated in seawater and freshwater

Mucosal and serum antibody responses were studied in sibling barramundi (Lates calcarifer) acclimated in either seawater or freshwater following vaccination by intraperitoneal injection or direct immersion in an inactivated Streptococcus iniae vaccine. As expected, route of vaccination had a marked effect on immune response, with direct immersion resulting in low serum antibody levels against S. iniae by ELISA detected 21 days post vaccination at 26 degrees C, whilst a significant response was detected in mucus. A strong specific antibody response was detected in both mucus and serum 21 days following intraperitoneal injection. Fish acclimated in seawater prior to vaccination showed a markedly higher specific mucosal antibody response than sibling fish acclimated in freshwater, regardless of the route of vaccination, whilst the serum antibody response was not affected by salinity. Both mucosal and serum antibodies from fish in seawater and freshwater were capable of binding antigen at salinities similar to full strength seawater in a modified ELISA assay. These results indicate that this euryhaline fish species is not only able to mount significant specific antibody response in cutaneous mucus, but that these antibodies will function in the marine environment.     

Slight influence of the estrous cycle stage on the mucosal and systemic specific antibody response induced after vaginal and intraperitoneal immunization with protoxin Cry1Ac from Bacillus thuringiensis in mice

Since the immune response appears to be variable according to the hormonal stage of the mammalian female, the aim of this study was to determine whether estrous cycle stage modifies the mucosal and systemic immune responses induced by intraperitoneal and vaginal immunization of mice with protoxin Cry1Ac. We tested the influence of three immunizations on the specific antibody response elicited at estrus and diestrus, that were the same estrous cycle stages at which the antigen was applied. Both intraperitoneal and vaginal immunization of mice with Cry1Ac either at estrus or diestrus induces specific antibody responses at serum, vagina and large intestine. The stage of the estrous cycle have little or non influence in the magnitude of the response induced, since only at serum the IgM was slightly higher at estrus than at diestrus by both routes. At the large intestine only the IgA response elicited via the intraperitoneal route changed, being higher at diestrus, whereas at the vagina IgA response induced did not change significantly due to the cycle stage. Present results suggest that Cry1Ac may be used as an antigen carrier as it can elicit antibody responses at systemic level and at several mucosal sites including the vagina that are not modified significantly throughout the reproductive cycle.     

Transfer Agent of Immunity

An immune ribonucleic acid (RNA) preparation was extracted with phenol from the spleens of guinea pigs immunized with diphtheria toxoid. Antibody-carrying cells were detected by immunocyte adhesion as rosette-forming cells. When germ-free rats, conventional guinea pigs or mice were injected intraperitoneally with this preparation, the rosette-formers were detected in either peritoneal exudate cells or spleen cells, whereas serum antibodies were unable to be detected thus far in such animals. Two injections with this preparation did not cause any remarkable increase in the number of rosette-formers, and serum antibody was also not detectable. By contrast, a high titer of serum antibody was demonstrated and the number of rosette-formers increased shortly after an injection of a small amount of diphtheria toxoid into guinea pigs which had previously received an injection with immune RNA. This reaction indicates a secondary response of antibody formation. However, secondary responses were not induced by injections of immune RNA preparations in guinea pigs primed with either diphtheria toxoid or immune RNA preparation. These facts suggest that immune RNA preparations did not contain antigens or fragments thereof and the immune response induced by RNA preparation is not the same as that induced by stimulation by the antigen itself. These results moreover can be accounted for by the notion that the immune RNA preparation is able to induce “memory” cells capable of responding to a secondary stimulus with an antigen and producing a high titer of serum antibody.     

IgE antibody-mediated cutaneous basophil hypersensitivity reactions in guinea pigs

Cutaneous basophil hypersensitivity (CBH) reactions are a heterogeneous group of delayed time course basophil-rich immune responses that can be mediated in the guinea pig by T cells, B cells, or IgG1 antibody. This study examined whether guinea pig IgE antibody could also mediate CBH reactions. IgE antibody to picryl or oxazolone determinants was induced by immunizing Hartley strain guinea pigs pretreated with cyclophosphamide. Hyperimmune serum from these animals was passed through a heavy chain-specific anti-IgG1 affinity column. The presence of IgE anti-hapten antibody in the filtrate fraction was verified by passive cutaneous anaphylaxis (PCA) testing with a 7-day period of local passive sensitization and by the heat lability (56 degrees C, 4 hr) of PCA activity. This IgE-rich fraction and the IgG1 fraction eluted from the column with base (0.2 M Na2CO3, pH 11.3) were transferred i.v. to separate groups of normal guinea pigs. Both fractions mediated delayed time course reactions that contained basophils. Macroscopic and microscopic reactions mediated by the IgE-rich fraction were abolished with heat (56 degrees C, 4 hr). Thus, two antigen-specific factors in guinea pig serum can mediate delayed time course basophil-containing reactions: IgG1 and IgE antibodies. IgE-mediated CBH reactions are similar to the late-phase reaction that follows IgE-dependent wheal-and-flare reactions in humans. The finding that guinea pig IgE can mediate a late reaction that contains basophils makes this a possible model for the human late-phase response, and suggests that some forms of CBH may play a role in human allergic disease.     

Antibody specificities generated during antigen-induced polyclonal hyperimmunoglobulinemia.

Much of the immunoglobulin produced during an immune response does not react with the eliciting antigen(s). The studies reported here were carried out to determine the antibody specificity of this immunoglobulin. Mice injected with antigen A displayed an increase in serum antibody to a previously experienced antigen (B). In addition, the spleens from such mice contained more antibody-forming cells directed against antigen B than did saline-injected controls. Thus, at least some of the immunoglobulin produced during the polyclonal hyperimmunoglobulinemia (PHIg) response is directed at antigens already experienced by the animal. In other studies, mouse serum reacted with autologous red cells treated with the proteolytic enzyme bromelain; the titer of this activity increased following immunization with several antigens and paralleled the PHIg response elicited by the same antigen. These results indicate that the antigen-induced PHIg contains antibody activity to autoantibodies.     

Transition in the Character of Immunological Memory in Mice after Immunization

The immunological memory in antibody response of mice to bovine serum albumin was investigated at the level of IgM and IgG antibody-forming cells. The antigen at a dose much lower than required for eliciting a detectable level of the primary antibody response could latently activate the immune machinery to an extent adequate for specific recall, whereas higher doses of antigen were effective in evoking strong anamnestic response. The potentiality to develop the anamnestic response was found even in the latent phase of the primary antibody response and was maintained for more than 2 months. The immunological memory acquired in an early phase after the primary immunization mainly involved IgM antibody response and late memory concerned IgG response.     

Immunopotentiation of the humoral response by liposomes: effect of a T cell polyclonal activator

In this paper, we analyzed the influence of surface-linked Con A on the secondary response to liposome-associated antigen via either encapsulation or covalent linkage at the liposomal surface. The study was carried out on BALB/c mice using bovine serum albumin as antigen. The humoral response was evaluated by measurements of antibody-producing cells (total, IgM, and IgG) and serum antibody titers. The results indicate that Con A at submitogenic concentrations does not potentiate the overall effect of liposomes but drastically changes the isotype distribution pattern obtained in response to encapsulated antigen without however affecting that obtained in response to surface-linked antigen. In all situations where Con A and/or BSA was covalently linked, IgG and IgM isotypes were produced in equal quantity, while in response to encapsulated BSA, IgG was by far the dominant isotype produced as expected for a thymo-dependent antigen. These results suggest that the quality of an immune response and the mechanisms of activation may be profoundly influenced by the nature of antigen association with liposomes as well as by the presence at the liposomal surface of immunomodulators such as Con A.     

Immunoregulation by antigen/antibody complexes. I. Specific immunosuppression induced in vivo with immune complexes formed in antibody excess

Specific immune complexes, prepared at different ratios of antibody to antigen, were examined for their effects on the antibody response of BALB/c mice to the cell wall polysaccharide antigen (PnC) extracted from Streptococcus pneumonia R36a. Mice immunized with complexes formed in antigen excess developed a PnC-specific antibody response that was equivalent to that in mice injected with free antigen. On the other hand, mice injected with complexes formed in antibody excess developed very little PnC-specific antibody. Furthermore, administration of immune complexes (formed in antibody excess) resulted in suppression of the response to an immunogenic dose of PnC given concurrently or 1 day after injection of immune complexes but not when the antigen was given 1 day before injection of the immune complexes. Injections of free antibody (TEPC-15) also resulted in suppression of the response to antigenic challenge; however, suppression was greatest when the antibody was injected concurrently with the antigen, suggesting that the suppression was mediated through the formation of immune complexes in vivo. The suppression appears to be specific for the antigen (PnC), since in mice injected with TEPC-15/PnC complexes (formed in antibody excess) and challenged with PnC coupled to sheep RBC, only the response to PnC was suppressed.     

The inhibition of cutaneous basophil hypersensitivity reactions by a heterologous anti-guinea pig T cell serum.

Systemic treatment with a heterologous anti-T cell serum of guinea pigs immunized with EA in IFA markedly suppressed CBH reactivity to specific antigen and T cell mitogens, as judged by gross reactivity, histology, and skin histamine. The antiserum produced a marked drop in circulating lymphocytes, mainly at the expense of T cells, as indicated by the ability of surviving lymphocytes to rosette with rabbit RBC. It was postulated that the suppression of CBH reactivity is due to the depletion of T cells, which would have released a factor chemotactic for basophils. The data therefore provide further evidence that cutaneous reactions rich in basophils are primarily dependent on a population of T cells.     

Antibody formation by one or both lymphoid populations present in chimeric marmosets

Development of an isoimmune serum capable of identifying a specific leukocyte antigen in the marmoset, Saguinus fuscicollis illigeri, permitted detection of lymphoid cell chimerism in this species by the cytotoxic test. This reagent was then used to identify the cell population in the chimera responsible for antibody production against a test antigen, sheep red blood cells. Primary in vitro antibody formation as measured by plaque-forming cells with blood leukocytes or splenic lymphocytes of six animals chimeric for the leukocyte antigen, MLA-1, revealed an immune response by both cell types of the chimeric population from three animals and a response by only one cell type in the other three.     

Verification of immune response optimality through cybernetic modeling

An immune response cascade that is T cell independent begins with the stimulation of virgin lymphocytes by antigen to differentiate into large lymphocytes. These immune cells can either replicate themselves or differentiate into plasma cells or memory cells. Plasma cells produce antibody at a specific rate up to two orders of magnitude greater than large lymphocytes. However, plasma cells have short life-spans and cannot replicate. Memory cells produce only surface antibody, but in the event of a subsequent infection by the same antigen, memory cells revert rapidly to large lymphocytes. Immunologic memory is maintained throughout the organism's lifetime. Many immunologists believe that the optimal response strategy calls for large lymphocytes to replicate first, then differentiate into plasma cells and when the antigen has been nearly eliminated, they form memory cells. A mathematical model incorporating the concept of cybernetics has been developed to study the optimality of the immune response. Derived from the matching law of microeconomics, cybernetic variables control the allocation of large lymphocytes to maximize the instantaneous antibody production rate at any time during the response in order to most efficiently inactivate the antigen. A mouse is selected as the model organism and bacteria as the replicating antigen. In addition to verifying the optimal switching strategy, results showing how the immune response is affected by antigen growth rate, initial antigen concentration, and the number of antibodies required to eliminate an antigen are included.     

Selective immunodepression

A variety of experimental conditions have been described which can selectively depress various aspects of the normal immune response. Treatment with cytotoxic drugs or variations in the route of administration or physical state of the antigen can selectively depress one immunoglobulin class while allowing normal synthesis of other immunoglobulin classes. Injection of excessive or subimmunogenic doses of antigen, or injection of antigen in a nonimmunogenic form will specifically depress antibody body syntheis to that antigen. Depending upon the antigen dose and other factors discussed above such tolerance can be selectively induced in either the B- or the T-lymphocyte population. Finally, antibody is highly heterogeneous with respect to its affinity for the antigenic determinant. As a consequence of the selective pressure of decreasing antigen concentration there is generally a progressive shift towards the production of high affinity antibodies. Various experimental maneuvers can selectively depress specific subpopulations of antibody forming cells. B-cell tolerance preferentially occurs in high affinity antibody forming cells with a decrease in the average affinity of the residual antibody formed. Passively injected antibody specifically depresses antibody synthesis to concomitantly injected antigen. This antibody mediated immune suppression selectively depresses low affinity antibody synthesis. Thus, a variety of experimental procedures have been discussed which will modify the immune response in highly selected ways.It is clearly important in describing the immune response to specify not merely the amount of antibody formed, but also its class and subclass. In addition, to fully describe the immune response it is necessary to specify the affinity and heterogeneity of the antibody. As discussed above the factors controlling the affinity of serum antibody and the mechanism of antibody mediated immune suppression are reasonably well understood. Much data are available regarding factors determining tolerance induction, however, the detailed cellular mechanisms involved remain obscure. With regard to the mechanisms determining which immunoglobulin classes are formed, and in what proportion, relatively little information is available.     

Plasmodium berghei: Reduction of the mouse's specific lymphoproliferative response in relation to corticosterone and pregnancy

Spleen cells from mice immune to Plasmodium berghei exhibited a significantly increased in vitro proliferative response to parasitized reticulocytes compared to spleen cells from normal mice. The specific response to malaria antigen was decreased in spleen cells from pregnant immune mice in contrast to the nonspecific response to the mitogen phytohemagglutinin. Addition of mouse serum to spleen cell cultures of immune mice depressed both the phytohemagglutinin and the specific proliferative response, whereas serum of pregnant mice exerted an even stronger inhibition than serum of nonpregnant mice. Charcoal adsorption of mouse sera for the elimination of steroid hormones removed the serum dependent immunosuppression from normal as well as pregnant serum. Corticosterone added to the spleen cell cultures depressed also the proliferative response. These findings demonstrate that the response to malaria antigen is decreased in immune mice during pregnancy. The possible effect of serum corticosterone on the depression of the immune response is discussed.     

Role of immune complexes in the humoral leukocyte adherence inhibition test

Summary The humoral leukocyte adherence inhibition (H-LAI) assay has recently been found to measure an antitumor immunefactor. In this assay, trypsinized leukocytes from control persons are used as indicator cells and 0,25% serum from the patient is added to the assay system together with the relevant tumor antigen.In the present work, evidence is presented that the H-LAI response is mediated through in vitro-formed immune complexes. Different antibody-antigen pairs (anti-albumin — albumin; anti-₂microglobulin — ₂microglobulin; anti-carcinoembryonic antigen — carcinoembryonic antigen; anti-transferrin — transferrin) have been added to the assay mixture. A significant H-LAI response was observed when immune complexes were formed. On the other hand, when unrelated antibody — antigen pairs were added, no response was found. The specificity was demonstrated in experiments where two different antibodies were added simultaneously and the response tested both against the two corresponding antigens and against unrelated antigens.Since the same trypsinized indicator cells can be used for different immune complexes, it is likely that the response is mediated through common receptors on the cell surface with affinity for immune complexes, i.e., Fc-receptors. Presumably, the H-LAI test gives response to immune complexes in general and is as such not specific. The specificity is achieved through the addition of specific antigen and the subsequent in vitro formation of immune complexes.     

Popliteal lymph node (PLN) assay to study adjuvant effects on respiratory allergy

Different variants of the popliteal lymph node (PLN) assay have been published. Here we describe the adjuvant popliteal lymph node assay, an immune response assay to study the adjuvant activity of soluble substances as well as particulate matter. The substance to be studied for adjuvant activity is injected into the hind footpad of mice or rats together with an antigen. Adjuvant activity is determined as the increase in PLN weight and cell numbers in animals receiving antigen together with the substance under study, compared with PLN weight and cell numbers in animals given the antigen without the substance in question, and animals given the putative adjuvant alone. Because lymph node weight and cell numbers are immunologically non-specific parameters, specific immune response assays like serum antibody responses or antibody-forming cell numbers should additionally be performed. Different antigens and immune response assays may be used, depending on the research question asked. In relation to respiratory (or food) allergy, the assays should as a minimum include determination of specific IgE in serum, and preferably also IgG1 (mouse). Serum specific IgG2a antibody determination may be added to get an indication of the Th1-Th2-balance of the response. The adjuvant PLN assay, with cellular response assays performed in the draining popliteal lymph node and antibody determinations in serum, requires small amounts of test material. The assay offers a practical, sensitive and reproducible method to determine the adjuvant activity of soluble substances as well as particulate material, with the possibility to also perform mechanistic studies.