Allotypic suppression of adult mouse spleen cells

Adult mouse spleen cells were transferred into irradiated adult mice with xenogeneic erythrocytes. Antibody-producing cells were measured by the localized haemolysis in gel (LHG) assay. Anti-allotype serum directed against the γG_2a allotype of the donor cells, given to the recipients at the same time as antigen and spleen cells, suppressed the plaque-forming cells (PFC) producing antibody of that allotype, and had little effect on the other classes investigated.     

Natural hemolytic activity of snake serum III. Plaque-forming cells in adult and newly-hatched snakes (Elaphe quadrivirgata)

The number and localization of cells secreting natural antibodies against sheep erythrocytes (SRBC) or rabbit erythrocytes (RRBC) were assessed in adult and newly-hatched striped snakes (Elaphe quadrivirgata) by a localized hemolysis in gel technique. In adult snakes, antibody-secreting cells, i.e. plaque-forming cells (PFC), were detected in the spleen, peripheral blood and thymus but not in the liver. The number of PFC showed great individual variations. The mean number of PFC against SRBC in the spleen, blood or thymus was about twice as much as that against RRBC. In newly-hatched snakes, PFC to both erythrocytes were detected predominantly in the liver, but scarcely in the spleen or thymus. The level of natural hemolysin in the serum and that of PFC in the liver against RRBC was higher than against SRBC. PFC to SRBC were distinct from those to RRBC. These studies are important for understanding both the ontogeny and phylogeny of natural antibodies.     

Antibody production studied by means of the localized haemolysis in gel (LHG) assay: II. Assay procedure

The localized haemolysis in gel (LHG or plaque) assay has been investigated and it has been shown that the best condition for the assay is a plate made up from a standard tissue culture medium (199) in an agarose gel. Several convenient but sub-optimal alternatives are described. Other variables that were investigated were the concentration of indicator erythrocytes in the assay plate, the amount of complement added, the incubation time and the neutralization of xenogeneic developing serum by free mouse serum γ-globulins. The reasons for using log(x+1) transformed data for statistical analysis are also presented.     

Enzymatic modification of lymphocyte receptors for antigen. III. Resistance of receptors to trypsin at the peak of the immune response

Using sheep erythrocytes (SRBC) as the antigen, two subpopulations of spleen antigen-binding lymphocytes could be distinguished by a marked difference in the susceptibility of their receptors to trypsin. In unimmunized animals, 30 % of the antigen-binding cells were trypsin-resistant, whereas at 5 days after immunization, 80–90% were trypsin-resistant, indicating an increase of about 50-fold in trypsin-resistant antigen-binding cells per spleen. In contrast, trypsinsensitive cells per spleen were only 4-fold higher on day 5 than before immunization. Therise in % trypsin sensitivity preceded the increase in rosettes per spleen, implying that immunization produced a preferential increase in trypsinresistant antigen binding cells partly by converting sensitive cells to resistant cells. After the 5th day, the trypsin sensitivity of antigen-binding cells slowly returned toward the unimmunized level, but a booster injection of SRBC restored trypsin resistance. Trypsin resistance was not lost in the presence of sodium azide or protein synthesis inhibitors. But a slightly increased trypsin susceptibility was conferred by 2-deoxyglucose, implying that glycolysis or the glycosylation of protein may be involved in maintaining trypsin resistance.     

Environmental modulation of lipopolysaccharide chain length alters the sensitivity of Escherichia coli to the neutrophil bactericidal/permeability-increasing protein.

The plaque-forming cell (PFC) assay with sheep erythrocytes (SRBC) sensitized with different antigens and a 4-h tritiated thymidine pulse assay were used to determine whether polyclonal activation occurs in rats following in vivo administration of Mycoplasma pulmonis. Injection of M. pulmonis into F344 rats resulted in an increase in the number of splenic immunoglobulin M-secreting PFC that produced antibodies reactive with the trinitrophenyl hapten and with SRBC. This polyclonal response reached a peak by 72 h after injection and returned to normal levels by 96 h, at which time the specific response to M. pulmonis reached its peak. Heat treatment and preopsonization of M. pulmonis with antiserum before injection resulted in reduced numbers of PFC against M. pulmonis-sensitized SRBC, trinitrophenyl hapten-sensitized SRBC, and SRBC. The number of PFC against the three types of target cells also increased in LEW rats after immunization with M. pulmonis. The number of PFC against SRBC and staphylococcal protein A-sensitized SRBC was higher in immunized LEW rats than in immunized F344 rats. Examination of unimmunized animals also revealed that LEW rats had higher initial numbers of PFC than did F344 rats. These results showed that polyclonal activation occurs in rats following in vivo administration of M. pulmonis and that LEW rats have an inherent propensity to develop higher nonspecific responses in vivo than F344 rats.     

Direct serological assay for the heat-labile enterotoxin of Escherichia coli, using passive immune hemolysis.

Sheep erythrocytes sensitized with the heat-labile enterotoxin (LT) of Escherichia coli exhibited passive immune hemolysis (PIH) when exposed to specific antitoxin and complement. Thus, PIH serves as the basis for an in vitro serological assay for LT that is sufficiently specific and sensitive to differentiate LT-positive and LT-negative E. coli isolates. The PIH assay for E. coli LT has been performed with the standard Microtiter system and also by a tube method employing the spectrophotometric determination of hemoglobin release. The spectrophotometric method enhances the sensitivity, accuracy, and objectivity of the PIH assay. The increased sensitivity of the spectrophotometric method also facilitates the identification of LT-positive cultures employing polymyxin "mini-extracts" of whole overnight (18 h) broth cultures of 2.0% Casamino Acid-0.6% yeast extract-salts medium rather than mini-extracts of cells derived from 3.5-h subcultures. Thus, large numbers of E. coli isolates can be individually tested for LT in less than 24 h after broth inoculation by a rapid in vitro assay which requires anti-LT serum as the only specific reagent.     

Immunochemical studies of organ and tumor lipids XXI. Sensitivity and specificity of the cytolipin F - sheep erythrocyte system.

The sensitivity and specificity of the sheep erythrocyte - anti-sheep erythrocyte system to inhibition by pure cytolipin F has been studied with 5 antisera, in order to compare it with the rat erythrocyte-anti-rat lymphosarcoma system and its inhibition by pure cytolipin R. The cytolipin F - sheep erythrocyte system is much more sensitive than the cytolipin R - rat erythrocyte system, inhibition of hemolysis of 6 x 10(6) sheep cells being produced by 10 ng of cytolipin F (combined with a four-fold quantity of lecithin) compared with inhibition of hemolysis of 10(6) rat cells by 50 to 100 ng of cytolipin R (also combined with lecithin). Differences in sensitivity are attributed to the larger number of available cytolipin F determinants on sheep erythrocytes compared with cytolipin R determinants on rat erythrocytes. Studies of the auxiliary lipid enhancement of cytolipin F activity by galactocerebroside, lactosyl ceramide (cytolipin H), and lecithin are also reported.     

Coupling of foot-and-mouth disease virus to sheep red blood cells using tannic acid for immunological assays

A technique for coupling foot-and-mouth disease virus (FMDV) to tanned sheep red blood cells (SRBC) is reported. Different parameters influencing the procedure were studied. Subtypes C₂, C₃, O₁ and A₂₄ were used as antigens, and guinea pig hyperimmune sera obtained were tested for specific antibody in passive hemagglutination (PH), passive hemagglutination inhibition (PHI) and passive immune hemolysis (PIL) assays. Fresh and SRBC stored in Alsever's solution showed similar behavior when used as indicator cells. Optimal sensitization of erythrocytes was achieved using tannic acid 1:20000 and 20 μg of purified virus/ml at pH 7.6. Specificity of the reaction was confirmed by PH and PHI in homologous and heterologous systems. The coupled antigen-antibody complex was sensitive to complement mediated lysis in a PIL test.     

Immunochemical Studies of Organ and Tumor Lipids Xxi. Sensitivity and Specificity of the Cytolipin F – Sheep Erythrocyte System

The sensitivity and specificity of the sheep erythrocyte – anti–sheep erythrocyte system to inhibition by pure cytolipin F has been studied with 5 antisera, in order to compare it with the rat erythrocyte–anti–rat lymphosarcoma system and its inhibition by pure cytolipin R. The cytolipin F – sheep erythrocyte system is much more sensitive than the cytolipin R–rat erythrocyte system, inhibition of hemolysis of 6x 10⁶ sheep cells being produced by 10 ng of cytolipin F (combined with a four–fold quantity of lecithin) compared with inhibition of hemolysis of 10⁶ rat cells by 50 to 100 ng of cytolipin R (also combined with lecithin). Differences in sensitivity are attributed to the larger number of available cytolipin F determinants on sheep erythrocytes compared with cytolipin R determinants on rat erythrocytes. Studies of the auxiliary lipid enhancement of cytolipin F activity by galacto–cerebroside, lactosyl ceramide (cytolipin H), and lecithin are also reported.     

Fc Receptor-Bearing Lymphoid Cells in the Chicken I. Characterization of the Fc(IgG) Receptor

The receptor for Fc(IgG) on chicken lymphoid cells was investigated by EA rosette techniques using sheep erythrocytes sensitized with a subagglutinating dose of anti-sheep erythrocyte (SRBC) chicken serum. Chicken lymphocytes did not form rosettes with SRBC coated with rabbit antibody, and human and mouse lymphocytes did not bind SRBC sensitized with chicken antibody. Only avian sera were effective in blocking the Fc receptor. Similarities between chicken and mammalian Fc receptors were demonstrated as both are pronase sensitive, trypsin resistant, and are distinct from surface immunoglobulin. Fc receptors were also distinguished from avian bursa-and thymus-specific antigens. Additional Fc receptor-bearing cells were revealed in bursa, spleen and bone marrow lymphocytes after neura-minidase treatment.     

Fc Receptor-Bearing Lymphoid Cells in the Chicken I. Characterization of the Fc(IgG) Receptor

The receptor for Fc(IgG) on chicken lymphoid cells was investigated by EA rosette techniques using sheep erythrocytes sensitized with a subagglutinating dose of anti-sheep erythrocyte (SRBC) chicken serum. Chicken lymphocytes did not form rosettes with SRBC coated with rabbit antibody, and human and mouse lymphocytes did not bind SRBC sensitized with chicken antibody. Only avian sera were effective in blocking the Fc receptor. Similarities between chicken and mammalian Fc receptors were demonstrated as both are pronase sensitive, trypsin resistant, and are distinct from surface immunoglobulin. Fc receptors were also distinguished from avian bursa-and thymus-specific antigens. Additional Fc receptor-bearing cells were revealed in bursa, spleen and bone marrow lymphocytes after neura-minidase treatment.     

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.     

A colorimetric-enzymatic microassay for the quantitation of antibody-dependent complement activation

In this report we describe a reliable, sensitive, safe, and easy way to assess antibody-dependent complement-mediated hemolysis. The assay is based on the quantitation of hemoglobin (Hb) released from lysed erythrocytes indirectly, through the generation of fluorene blue, a compound formed from 2-7 diaminofluorene in an enzymatic reaction catalyzed by the Hb molecule. The fact that Hb is the most abundant protein within a mature RBC (approximately 10(11) molecules per cell) and possesses pseudoperoxidase activity, makes the fluorene blue-coupled assay more sensitive than the simple estimation of Hb adsorption at 410 nm (Soret adsorption maxima of Hb), and as sensitive and reliable as its radioactive equivalent based on the release of 51Cr from previously loaded RBCs. Using this assay chimeric mouse-human anti-dansyl antibodies, comprising all the human IgG isotypes, were tested for their ability to mediate complement activation. The results obtained agreed with previously reported data, confirming that the fluorene blue-coupled assay is reliable. This assay also has significant advantages over the radioactive-based assay in that the reagents used are inexpensive, and the concerns of using radioactivity and the associated hazards are obviated. Because there is no need for loading the target cells with the analyte to be detected since RBCs are already loaded with Hb, the fluorene blue-coupled assay is simpler and eliminates many steps in comparison to the 51Cr release based assay.     

Two T lymphocyte populations as effector cells fro IgG dependent cytotoxicity.

The characteristics and proportions of human lymphocyte populations capable of lysing IgG coated target cells were studied. Monolayer of target erythrocytes coated with rabbit IgG were employed to detect and isolate the lytic effector lymphocytes. Normal peripheral blood effector lymphocytes were shown to express IgG receptors, and a total of four cell types possesing different surface markers were identified. An average of 68% (range 51-80%) of the effector lymphocytes had T lymphocytes antigen and were capable of SRBC rosette formation, and 32% of the lymphocytes lacked T lymphocyte markers. Two populations of these T lymphocytes were identified: one possesses SRBC and IgG receptors, and the other possesses SRBC, IgG and complement receptors. The proportion of the former was estimated as 53.2% and the latter as 14.8% of the total effector cell population. Two other cell types were distinguished among the non-T lymphocytes. One type of lymphocytes bear IgG receptors (21.6%) and the other has both IgG and complement receptors (10.4%). The cytolytic capacity of the two T lymphocyte subpopulations is suggested to be an important reaction of the immune reactive T lymphocytes during the immune response.     

Immunogenicity and antigenicity of synthetic Escherichia coli lipid A.

The immunogenicity and antigenicity of synthetic Escherichia coli lipid A (compound 506) and its 1- and 4'-monophosphorylated derivatives (compounds 505 and 504, respectively) and nonphosphorylated derivative (compound 503) were compared with those of bis- and 4'-monophosphorylated natural free lipid A from E. coli. The synthetic compounds under study were either coated onto sheep erythrocytes (except for the water-insoluble preparation 503) or incorporated into liposomes and used for the immunization of rabbits. Both types of immunogens (the latter representing fully synthetic immunogens) resulted in high-titered polyclonal antisera which were characterized before or after absorption in a passive hemolysis assay as well as in a passive hemolysis inhibition assay with the synthetic compounds as test antigens. All antisera were found to react with their corresponding homologous antigens coated onto sheep erythrocytes, with titers of up to 2,048, and were comparable to those antisera obtained after immunization with natural lipid A exposed on the bacterial surface after acid hydrolysis. Antisera against bisphosphorylated compound 506 were highly specific for the homologous antigens, showing no interaction with compounds 504 and 505 in the passive hemolysis test. The same held true for the absorption experiments in which glutaraldehyde-fixed sheep erythrocytes were sensitized with the respective antigens. Antisera against monophosphorylated compounds 504 and 505 exhibited, besides their expected homologous reactivity, complete cross-reactivity with compound 506, but they did not cross-react with each other. Thus, anti-504 and anti-505 antibodies recognized distinct antigenic determinants, being related to the ester linked 4'-phosphate or the glycosidically linked 1-phosphate, respectively. Both antigenic determinants were also expressed by bisphosphorylated compound 506 used as an antigen; however, upon immunization, only antibodies against compound 506 were elicited.     

The complement system of the duck.

Antibody (Ab)-dependent and-independent activation of the duck complement (C') system were studied. Ab-independent C' activity exhibited characteristics similar to those of the mammalian alternative C' pathway (ACP), including the selective lysis of rabbit erythrocytes (RRBC), a requirement for Mg2+, but not Ca2+, depletion of activity by zymosan, and lack of sensitivity to the mammalian C1 inhibitor carrageenan. Measurement of C' activity using antisera against sheep erythrocytes (SRBC) revealed that duck Abs activate C' by a pathway resembling the mammalian classical pathway (CCP) requiring both Ca2+ and Mg2+. Ab-dependent and-independent activities were further distinguishable by their kinetics of lysis and sensitivities to heat. Duck Abs were also found to activate C' in normal and carrageenan-treated serum by a mechanism that requires only Mg2+, and thus resembles the ACP. However, this Ab-dependent ACP-like activity exhibits patterns of ionic strength dependence and ontogeny which are clearly different from those of the conventional ACP and CCP. These findings indicate that duck C' can be activated by three mechanisms: Ab-mediated activation of the CCP, and Ab-mediated and Ab-independent activation of the ACP. Duck Ab responses to SRBC and RRBC were followed by direct agglutination, antiglobulin agglutination, and activation of the CCP and ACP. While the C'-activating abilities of duck anti-SRBC Abs persisted through a 3-month programme of inoculation, the anti-RRBC response lost its ability to activate C' after 2 weeks.     

The C8-binding protein of human erythrocytes: interaction with the components of the complement-attack phase.

C8-binding protein is an intrinsic membrane protein of the human erythrocyte. It inhibits the complement (C5b-9)-mediated lysis in a species-restricting manner. In the present study we incorporated C8bp, isolated from human erythrocytes, into sheep erythrocytes (SRBC). SRBC, normally sensitive to lysis by human C5b-9, became insensitive to lysis. Furthermore, we found that C8bp is incorporated into the membrane-attack complex C5b-9, most probably by interacting with C8, since C8bp has an affinity for C8, particularly for the C8 alpha-gamma-subunit. Antibodies to C8bp react with the C8 alpha-subunits and with C9, pointing to the possibility of a partial homology between these proteins.     

Non-Determinant Specificity of Feedback Immunosuppression by IgG Antibodies Injected after the Antigen

The determinant specificity of the IgG-mediated suppression of the humoral immune response in mice was studied. One hour before or 2.5, 6, 12, or 24 h after the injection of sheep erythrocytes (SRBC) or SRBC-TNP, CBA/Ca mice received SRBC-specific monoclonal IgG antibodies. The antibodies did not cross-react with TNP or goat erythrocytes, the latter an antigen which shows 30% cross-reactivity with SRBC. Five days later the determinant-specific plaque-forming cell response against SRBC and the non-determinant-specific response against goat erythrocytes and TNP were determined. Regardless of whether the antibodies were injected before or after the antigen, they suppressed not only the response to the antigenic determinant they bound to, but also the response to other epitopes on the same antigen. This shows that Fc parts of the IgG molecules play a crucial part in suppression of the in vivo antibody response even when, as in a natural situation, the antigen is presented to the immune system before the antibody.     

Specific suppression of delayed hypersensitivity response to sheep erythrocytes by heterologous anti-lymphocyte serum.

The development of a heterologous anti-lymphocyte serum (ALS) capable of specifically suppressing the delayed hypersensitivity (DH) response is reported. This ALS, termed ALS(CMI), was prepared against lymph node cells from rats which had been immunized against sheep erythrocytes (SRBC) following treatment with cyclophosphamide which is known to enhance the DH response and suppress the humoral immune response. The effect of ALS(CMI) on the primary DH response to SRBC using the footpad swelling test was studied. Its effect on the primary humoral immune response to SRBC was also studied using the Jerne plaque assay technique. ALS(CMI) suppressed the humoral antibody response to SRBC and the DH response to a third party antigen only when administered before the antigen, having no effect when administered post-antigenically. On the other hand, ALS(CMI) significantly suppressed the primary DH response to SRBC when administered either before or after the antigen.