Direct and indirect plaque forming cells in extrapulmonary lymphoid tissue following local vs systemic injection of soluble antigen

AKR strain mice were immunized with solubilized SRBC stroma either by direct injection into the lower respiratory tract or intravenously via the tail vein. The number of plaque forming cells (PFC) in the draining plumonary lymph node (tracheobronchial node) and spleen were determined by direct (IgM) and indirect IgG₁, IgG_2b, IgA) plaque assays.Intravenously administered antigen induced an initially strong IgM response in the spleen which was subsequently followed by antibody of the IgG₁, IgG_2b, and IgA classes of immunoglobulins. The tracheobronchial lymph node contained a minimal number PFC representing all four types of immunoglobulins studied. Conversely, following a single local injection of antigen directly into the lower respiratory tract, the tracheobronchial node responded with relatively high concentrations of PFC of all classes. The response in the spleen, although higher than background, was barely detectable. The splenic response to locally administered antigen was, however, considerably augmented as a result of a second local injection given 45 days after the initial stimulation. Under these conditions, IgG₁ IgG_2b, and IgA were represented in both tissue sites by sharp increases in the number and a decrease in the time of appearance of their respective antibody forming cells. Comparable changes were not noted for the case of IgM.Serum hemagglutination titres following a single injection by either route did not vary significantly during the time course of the experiment (28 days). The sera from locally immunized mice were treated with the reducing agent dithiothreitol and hemagglutination titres, before and after treatment, were compared. The major serum activity observed during the first 10 days following injection was affected by reduction and could therefore be assigned to high molecular weight antibody (19S, 13S). Subsequent titres (Days 13–26) were less susceptible to DTT and are considered to represent low molecular weight immunoglobulins (7S).     

In vitro inhibition of the helper activity of GAT-specific T-cell lines by a syngeneic anti-idiotypic serum: preferential effect on the IgG response

We described in this paper the characteristics of a syngeneic anti-idiotypic serum made in BALB/c against BALB/c anti-poly (Glu⁶⁰ Ala³⁰ Tyr¹⁰) (GAT) antibodies. This serum recognizes idiotypic determinants present in all anti-GAT sera whatever the allotypic markers of the mice used to prepare the sera. The functional effect of this serum on two helper cell lines is also described. Cell line BDF₁/52 was obtained from GAT immunized lymph node cells (LNC). Cell line BDF₁/E₃ was selected from splenic T-cells educated in vitro on GAT-pulsed adherent cells. Both lines were propagated in presence of filler cells, antigen, and medium containing T-cell growth factor(s) from splenic cells activated with concanavalin A. Both cell lines exhibit a helper activity as measured by the plaque-forming cell (PFC) response they induce in vitro in the presence of DNP-GAT and DNP sensitized B cells. Their helper activity is specific and they require a hapten-carrier bridge to activate B cells. These lines are able to induce IgG₁, IgG_2a and IgG_2b anti-TNP PFC. Syngeneic anti-idiotypic serum B 658 inhibits specifically the function of these two lines but does not affect the helper activity of an OVA-specific T-cell line. The blocking activity of the serum can be adsorbed on a hybridoma protein with anti-GAT activity. This inhibition affects more dramatically the IgG₁ response than the IgG_2a and IgG_2b responses.     

Secretion of IgM antibodies by glass-adherent fraction of immune mouse spleen cells. II. Characteristics of glass-adherent plaque-forming cells.

Further studies have been carried out on the nature and the behaviour of the anti-SRC plaque-forming cells from the adherent fraction of mouse spleen cells. In this fraction many phagocytic, acid phosphatase-positive cells were observed in the center of the plaques. The glass-adherent PFC were found to be highly radioresistant in vitro, compared to the nonadherent fraction. In both primary and secondary immune responses, only the direct PFC showed the tendency to adhere to glass. The phenomenon of the glass adherence of a fraction of direct PFC is also apparent in mouse spleen cells stimulated in vitro with SRC.     

Modulation of mouse anti-SRBC antibody response by placental extracts

Mouse placental extracts (PE) and corresponding Sephadex G-200 fractions were administered to isogeneic CBA mice along with an optimal immunizing dose of SRBC. Spleen cells were harvested 8 days later and transferred to CBA recipients, subsequently immunized with SRBC. The immunoregulatory activity of spleen cells from PE-treated donors was compared to cells from liver extract (LE)-treated controls or from mice immunized with SRBC only, using Cunningham's PFC direct and indirect tests. Within the dose range used, selective modulatory activities were obtained with cells from PE, but not from LE, treated mice, the latter being comparable to cell transfer effects from donors immunized with SRBC only. Spleen cells from animals injected with low doses of PE (0.25 to 4 mg per mouse) added to immunizing SRBC had a suppressive effect on the primary IgM response of recipients immunized against SRBC. In contrast, when SRBC were given to donor animals with higher doses of PE (8 to 13 mg), transferred spleen cells potentiated the IgM response of the recipients. These opposite suppressive and potentiating activities were found in distinct Sephadex G-200 fractions of 40 and 60 kDa, respectively. When the effect of PE treatment was tested within the same animal, the indirect secondary PFC response following a challenge with SRBC was significantly modified. We observed an overall suppression of the different isotypes after treatment with lower doses of PE or with its 40-kDa fraction. PE doses of 0.5 to 2 mg resulted in a stronger inhibition of IgM than IgG₁ production. This phenomenon was also obtained with the 40 KDa fraction. IgG₂ responses were significantly reduced by all doses of this fraction. In contrast, all doses of the 60-kDa fraction gave a strong stimulation of IgG₂ and IgM responses and a constant suppression of the IgG₁ response. This shows a clear dissociation between IgG₁ and C'-fixing (IgM, IgG₂) antibody classes as far as the influence of placental substances is concerned in their regulation. These data emphasize the relevance of isogeneic placental products as a useful physiological material capable of modulating xenogeneic immune responses (as well as allogeneic systems).     

Acetylcholinesterase secretion by parasitic nematodes. IV. Antibodies against the enzyme in Trichostrongylus colubriformis infected sheep

Sheep infected with the nematode parasite Trichostrongylus colubriformis showed anti-T. colubriformis acetylcholinesterase. (AChE) antibodies in the IgG₁ but not the IgG₂ or IgM fractions prepared from their serum. Using the fluorescent antibody technique with representative sera, antibodies in the IgG₁ fraction exhibited specificity for antigens in the subventral glands of the worm excretory system. IgA antibody specificity for antigens in the excretory glands and intestine of the worm was also demonstrated.     

Antibody formation in mouse bone marrow. VII. Evidence against the migration of plaque-forming cells as the underlying cause for bone marrow plaque-forming cell activity: a study with parabiotic mice

After intravenous immunization of mice with Escherichia coli lipopolysaccharide (LPS) or sheep red blood cells (SRBC), the bone marrow can contain large numbers of plaque-forming cells (PFC). By means of parabiosis, it was studied whether or not this appearance of PFC in the bone marrow might be due to a migration of such cells from peripheral lymphoid organs into the marrow, as has been suggested in the literature. Using parabionts consisting of nonimmunized mice and mice immunized with LPS, only background numbers of PFC could be demonstrated in the bone marrow of the nonimmunized mice. In similar experiments, with SRBC as antigen, mice showing high anti-SRBC PFC activity in the bone marrow could only provide for minor numbers of anti-SRBC PFC in the bone marrow of affixed normal mice. These results suggest that migration of PFC can not be the main cause for bone marrow PFC activity in the mouse. This provides additional evidence for our view presented in previous papers of this series that the appearance of PFC activity in the bone marrow is dependent on local maturation of B cells into PFC rather than on immigration of PFC.     

Activation of human B lymphocytes: XVI. Cellular requirements,interactions, and immunoregulation of pokeweed mitogen-induced total-immunoglobulin producing plaque-forming cells in peripheral blood

The optimal culture and assay conditions for the detection of spontaneously occurring and pokeweed mitogen (PWM)-induced polyvalent Ig (IgG + IgM + IgA) and individual Ig class-specific plaque-forming cells (PFC) in human peripheral blood have been described in detail. Culture conditions are critical, particularly with regard to cell density and batches of supplemental serum. Fetal calf serum is a much more supportive serum supplement for PWM-induced PFC than is human serum. The assay system is a modified reverse hemolytic PFC assay using staphylococcal protein A coupled to sheep red blood cells by the chromic chloride method. PFC are developed by rabbit anti-human polyvalent Ig or anti-human individual Ig class antisera. Human peripheral blood contains 468 (±78) spontaneously occurring Ig secreting PFC per 10⁶ lymphocytes at Day 0 and 20,500(± 1971) PWM-induced Ig secreting PFC after 6 days in culture. The response is T-cell dependent; however, T cells can be replaced by a soluble T-cell factor prepared from a 48-hr allogeneic mixed lymphocyte reaction supernatant. The relative dependence on monocytes is a reflection of the culture conditions employed. Under the conditions of round-bottom tubes which promote cell-to-cell contact, depletion of monocytes to 0 to 2% does not result in a diminution of PFC responses. In fact, under such conditions, in certain individuals monocytes are markedly suppressive such that removal of monocytes results in a substantial enhancement of PFC responses. This system is simple and reproducible and should prove extremely useful in the delineation of the mechanisms of B-cell triggering and immunoregulation in normals and in disease states.     

In vitro studies of the rabbit immune system: I. Hemolytic plaque-forming response of dissociated spleen cells from normal and primed rabbits

The conditions for the in vitro generation of primary and secondary immune responses by rabbit spleen cells to sheep red blood cell (SRBC) antigen have been examined. Spleen cells from many normal and all previously immunized rabbits are capable of producing in vitro plaque-forming cell (PFC) responses when cultured as dissociated cell suspensions in the presence of antigen. Primed spleen cells generate approximately 100 times the number of PFCs obtained in normal cultures with a shorter lag period. Both types of cultures demonstrate a period of exponential increase in PFCs during which the doubling time is 12–14 hr. This increase occurs after 1 day of culture of spleen cells from primed rabbits and after 4 days of culture of spleen cells from unprimed rabbits. The PFCs which arise in cultures of primed cells appear not to be the progeny of those generated in vivo but to be derived from an increased number of PFC precursors. Repeated immunization of the spleen cell donor is required to produce significant numbers of indirect (IgG) PFC or indirect precursors; most of the PFC found after a single immunization in vivo or in vitro are direct (IgM). There is no evidence for conversion of IgM to IgG PFC in vitro. This system should provide a means for further identification of the cellular interactions involved in the immune response of the rabbit.     

Experimental trichinosis-II. Quantitative and qualitative variations of serum in CBA mouse and Wistar rat (author's transl)]

Variations of serum globuline and immunoglobulins were measured by electrophoresis on cellulose acétate and radial immunodiffusion during the course of infection in inbred CBA mice and Wistar rats.In the two animal species, electrophoresis showed a significant decrease of serum albumin attending an increase of γ globulins. This phenomenon was earlier and greater in mice than in rats. This difference might be explained by suggesting that Trichinella is better adapted to rats than to mice.Beginning in the second week of infection, all classes of immunoglobulins were increased in the mouse with IgG_2a and IgA showing the greatest increase. The different classes of immunoglobulins continued to increase during the 4th month of infection but the rate of increase diminished after the second month except for IgA which increased between the 60th and 120th day.     

Variations in the responses of C57BL and a mice to sheep red blood cells: II. Analysis of plaque-forming cells

The number of direct and indirect plaque-forming cells (PFC) and the serum hemolytic activity was determined for A/He, C57BL/6J, and B6AF1 mice responding to multiple injections of sheep red blood cells (SRBC). Although the kinetics of the primary response differed, all mice had high numbers of both direct and indirect PFC and low-titered 2-mercaptoethanol (2-ME) sensitive serum antibody. Following multiple SRBC injections, the A/He spleens contained predominantly IgG producing PFC. Their serum antibody activity was resistant to 2-ME signifying the presence of IgG. The serum activity of both the C57BL/6J and B6AF1 mice was sensitive to 2-ME (IgM antibody) over the course of immunization, and although there was a definite IgM PFC memory response, the presence of 7S memory PFC was questionable. The results are discussed in terms of the maturation of the antibody response to SRBC and of the question of the postulated IgM and IgG switch.     

The absence of VPAC2 leads to aberrant antibody production in Aspergillus fumigatus sensitized and challenged mice

Vasoactive intestinal peptide (VIP) facilitates a “pro-allergy” phenotype when signaling through its G protein-coupled receptor, VPAC₂. We have shown that VPAC₂ knock-out (KO) mice developed an allergic phenotype marked by eosinophilia and elevated serum IgE. Therefore, we hypothesized that the humoral response to allergen challenge in these mice was T_H2 dominant similar to wild-type (WT) C57BL/6 mice. Antibody responses in WT and KO mice were measured after Aspergillus fumigatus conidia inhalation. In contrast to previous reports, basal levels of serum IgG_2a and IgA were significantly higher in naïve VPAC₂ KO animals. Antibody availability in the serum as well as the bronchoalveolar lavage fluid after fungal challenge was dominated by the pro-inflammatory isotype IgG_2a and the mucosal isotype, IgA. IgA localizing cells dominated in the peribronchovascular areas of allergic KO mice while IgE immune complexes were found in WT allergic lungs. This research shows for the first time that VPAC₂ has a significant effect on antibody regulation, in the context of allergy.     

Deregulation of mouse antibody-forming cells by streptococcal pyrogenic exotoxin II. Modification of spleen T-cell-complemented nude mouse PFC responses

Streptococcal pyrogenic exotoxin (SPE), a toxic protein, secreted by Group A streptococci modifies antibody responses in two ways. It suppresses the early peak plaque-forming cell (PFC) and serum antibody responses to sheep erythrocytes (SE) and it engenders a late burst of PFC detected at 12–14 days. We have termed the late phase a deregulated response. This effect has been observed in rabbits and NIH (+/+ and +/nu) mice. NIH athymic nude (nu/nu) mice exhibit the early suppressed response but do not show the late phase. In reconstruction experiments to delineate the responsible target site of SPE we have conferred upon the nude or nude spleen cells in vitro, +/nu PFC responsiveness to SE by transfer of +/nu spleen cells in vivo or by supplementation with +/nu spleen cells in Marbrook cultures. When this is done, complementation of nude PFC responses and their ability to exhibit a deregulated response after SPE treatment is conferred coordinately. Pretreatment of donor cells with a B-cell inhibitory dose of X-ray or with a B-cell inhibitory dose of anti-Ig serum + C′ does not inhibit complementation of nude cells to +/nu responsiveness. Moreover, such donor suspensions when treated with SPE retain the ability to complement and to confer upon nude cells the ability to exhibit the late burst of PFC (a deregulated response). A similar pretreatment of the donor cell suspension with an anti-T-cell serum and C′, however, markedly inhibits both the adoptive complementation and the deregulation of the nude mouse PFC response. Thus, it is demonstrated that the target cell affected in this way by SPE is a T-cell. We presume from this evidence that SPE inhibits a T-cell which is involved in the regulation of antibody formation.     

Heligmosomoides polygrus (equals nematospiroides dubius): humoral and intestinal immunologic responses to infection in mice

Measurements of serum IgG₁, IgG_2a, IgM, and IgA levels and antibody titers in these immunoglobulin classes were made at intervals after initial infection and challenge infection of mice immunized by two or three previous infections. Identical measurements were made on the content of the small intestine in mice which had been exposed to the same infection schedule. Sections of small intestine taken after initial infection and challenge infection were examined by the fluorescent antibody technique for changes in populations of immunoglobulin-containing cells and by routine histologic procedures for histopathologic changes.In serum, only IgG₁ was consistently increased after initial infection, and antibody in IgG₁ was detected within the first 2 wk of infection. In immunized animals, only IgG₁ and antibody of this class always responded to challenge infection, although antibody in other immunoglobulin classes was detected.IgA concentration of the intestinal content did not differ significantly after initial infection or challenge infection of immunized mice. Immunized mice had about twice the IgG₁ concentration in intestinal content as singly infected animals. No intestinal antibody was detected after initial infection; only IgG₁ antibody was detected in the intestinal content of immunized and challenged mice.Cell infiltrates in the intestinal mucosa and submucosa of immunized animals contained numerous IgG₁-containing cells. Mast cells and globular leukocytes were observed in the intestine of immunized animals.     

Reactivity of bacterial Fc receptors with bovine immunoglobulins: implications and limitations for quantitative immunochemistry

Summary The interaction of bovine immunoglobulins with staphylococcal Protein A and a group C streptococcal bacterial Fc receptor (FcRc) were compared. The isolated group C streptococcal receptor was reactive with both bovine IgG₁ and IgG₂. The reactivity of the streptococcal FcRc with IgG₂ was approximately 40 fold greater than that observed with IgG₁. By contrast, protein A reacted only poorly with bovine IgG₂ and no detectable reactivity was observed with IgG₁. A two stage competitive binding assay to measure bovine IgG in serum and secretions using ¹²⁵I-labeled protein A as tracer was developed. This assay was found to be sensitive and reproducible and was used to measure serum IgG levels in cattle of differing ages and breeds.     

The properties of plaque-forming cells from autoimmune and normal strains of mice with specificity for autologous erythrocyte antigens.

Treatment of mouse erythrocytes with the proteolytic enzymes, bromelain, reveals antigenic determinants not normally exposed on the erythrocyte surface. It was found that not only NZB mice, a known autoimmune strain, but also several normal strains of mice contain cells in small numbers in their spleens and in larger numbers in their peritoneal cavities which will form plaques against bromelain-treated MRBC. During in vitro culture the number of anti-BR-MRBC PFC increases slightly in the spleen cell populations whereas the number of these PFC in peritoneal cells increases dramatically to as many as 100,000 PFC/10(6) cells. The plaques detected in this assay contain a central lymphoid cell and their development, which requires the presence of complement and protein synthesis, is inhibited by anti-mouse immunoglobulin.     

Separation of mitogen-induced suppressor cells of human antibody-producing cells

Human antibody-forming cells were demonstrated by a plaque in agar technique following in vitro stimulation with either pokeweed mitogen or Cowan I strain of protein A-positive Staphylococcus aureus bacteria. We evaluated the effects on this antibody formation caused by the addition of cells which had been stimulated with PH A or Con A. Both Con A and PHA cells harvested after 3 days showed strong inhibition of pokeweed-induced plaque formation. The majority of the suppression could be accounted for by a blast fraction separated on 1g sedimentation gradients from the Con A or PHA cultures. Small cells from such cultures showed inhibition of PFC when added at high ratios (1:2), but this suppressive activity diluted out much more rapidly than that of the blast cells. No helper activity was noted with either small cells or blasts. Our studies indicate a T-cell blast as the suppressive fraction in Con A- or PHA-stimulated human lymphoid cells. While this T-cell suppression applies to T-dependent responses such as antibody stimulation with pokeweed mitogen, it does not have a substantial effect on Cowan I-induced plaque-forming responses. The finding that Cowan I-induced plaques could not be inhibited by Con A or PHA blasts indicates the T independence of this response.     

The Interactions of Porcine and Ovine,Serum and Colostral Immunoglobulins with Staphylococcal Protein A

The purpose of these studies was to determine the proportion of each immunoglobulin class/subclass in blood and colostrum of the pig and sheep, which would bind to staphylococcal Protein A. The concentrations of porcine IgG, IgM, and IgA were determined for serum and colostral whey from five sows. Similar measurements were made on two fractions produced by elution of the sample through a Protein A-Sepharose column: fraction 1, immunoglobulins which did not bind to Protein A, and fraction 2, immunoglobulins which bound to Protein A. The concentrations of ovine IgG₁, IgG₂, IgM, and IgA were measured for serum and colostral whey from six ewes, and again similar measurements were made after elution of each ovine sample through Protein A-Sepharose. All classes/subclasses of porcine and ovine serum and colostral immunoglobulins bound to Protein A to some extent. More than 90% of IgG from both porcine colostral whey and serum bound to Protein A. Ovine IgG₁ from most ewes possessed a low affinity for Protein A whereas ovine IgG₂ generally possessed a high affinity; 100% of the IgG₂ in ovine colostral whey samples bound to Protein A. There was remarkable variation between individuals in the binding capacity of porcine IgM and each of the ovine immunoglobulins. For the ovine samples, in particular there were distinct differences between Protein A binding capacity of serum and colostral immunoglobulins of the same class/subclass.     

Schistosoma mansoni: identification of immunoglobulins associated with the tegument of adult parasites from mice.

Immunocytochemical and immunodiffusion studies were conducted in an attempt to identify the immunoglobulins associated with the tegumental surfaces of Schistosoma mansoni. Peroxidase-labeled monospecific rabbit anti-mouse immunoglobulin class or subclass sera revealed the presence of mouse IgG₁, IgG₂, IgG₃, IgA, and IgM on the surface of adult parasites recovered from mice. These observations were confirmed by double gel diffusion of the various rabbit antisera against an eluate obtained from mouse worms.     

Apparent T cell function of bone marrow cells from mice experiencing a graft-versus-host reaction.

The graft-versus-host (GVH) reaction, induced in adult F₁ mice by the injection of parental strain lymphoid cells (GVH mice), suppressed the in vitro plaque-forming cell (PFC) response to sheep erythrocytes (SRBC) of spleen cells obtained from the GVH mice (GVH-SC). In vitro restoration of the PFC response of GVH-SC was carried out employing a modified Marbrook culture chamber consisting of an inner culture compartment (IC) separated from an outer culture compartment (OC) by a cell-impermeable membrane. Thymus cells (TC) and lymph node cells (LNC) but not bone marrow cells (BMC) from normal mice placed in the IC restored the PFC response of GVH-SC cultured with SRBC in the OC. The restoring ability of TC and LNC was markedly reduced following treatment with anti-theta serum plus complement. BMC taken from GVH mice 3 or more days post-GVH induction (GVHBMC) and placed in the IC restored the PFC response of GVH-SC as well as TC and LNC. Treatment of GVH-BMC with anti-theta serum plus complement did not affect their restoring ability; furthermore, the number of theta-bearing cells in the bone marrow did not increase as a consequence of the GVH reaction. Two possible explanations are proposed for the T-like function of GVH-BMC.     

RNA conversion of lymphoid cells to synthesize allogeneic immunoglobulins in vivo

Ribonucleic acid extracts (“5 day immune” and “nonimmune”-RNA) obtained from lymph nodes and spleens of rabbits homozygous for the b⁴ or b⁵ allele of light chain immunoglobulin allotypes were injected iv into nonimmunized rabbits homozygous for the alternate allele. The recipient rabbits were then given multiple iv injections of sheep red blood cells (SRBC). The spleens were assayed 13, 21, and 37 days following the RNA injection for “direct” IgM and “indirect” IgG plaque forming cells (PFC) specific for SRBC. The b4 or b5 light chain allotype and the a1, a2, and a3 heavy chain allotype of the antibody in the plaques was identified by radioautography and by inhibition of plaque formation using anti-allotype antibodies. The b light chain allotype of the RNA donor was identified in 22–32% of the IgM plaques and in 25–42% of the IgG plaques. The allotype of the host rabbit b light chain allotype was identified in 56–67% of the IgM plaques and in 57–71% of the IgG plaques. Likewise the a heavy chain allotype of the RNA donor was identified in 10–19% of the IgM plaques and in 12–19% of the IgG plaques. The allotype of the host rabbit a heavy chain allotype was identified in 51–60% of the IgM plaques and in 55–63% of the IgG plaques. The concentrated lysates of spleen and lymph node cells were also analyzed for immunoglobulins of each light chain allotype by immunodiffusion with radiolabeled antibody. The allotype of both the RNA donor rabbit and host rabbit were found in most of the lysates of lymphoid tissues and in some of the IgG isolated from the serum and concentrated.