Higher ADCC of murine peritoneal cells after immunization with allogenic tumor cells as compared with stimulation by adriamycin, BCG, and thioglycolate

Normal peritoneal cells or spleen cells from C57BL mice could not lyse SRBC in an ADCC assay. After intraperitoneal injection of Adriamycin, BCG or thioglycolate the ADCC of peritoneal cells toward antibody-coated SRBC was elevated to 30% in contrast to the ADCC of spleen cells. However, peritoneal cells but not spleen cells of mice immunized with allogenic tumor cells (DBA SL2) showed ADCC levels at least two times higher than the levels observed after stimulation by other agents. Maximal ADCC levels (55.8%) were observed 10 to 15 days after immunization. Direct cytotoxicity towards SRBC increased to a maximum of 17.7% at 9 days after immunization. The effector cells in this system are thought to be macrophages, for ADCC activity was only present in the plastic-adherent cell fraction. Cell to cell contact was necessary for ADCC to occur; nonsensitized erythrocytes were not lysed when added to a mixture of effector cells and sensitized erythrocytes. Concentrations of antibody of 1 pg/ml were sufficient to induce ADCC, and effector cell to target cell ratios could be as low as 0.05. The finding that macrophages of mice immunized with allogenic tumor cells exhibit higher ADCC levels than macrophages elicited in other ways can contribute to the investigation of combined cancer therapy with antibodies and biological response modifiers.     

The immunomodulating properties of erythrocytes induced by physical factors]

It is shown that the action of ultraviolet rays (UVR) or magnetic field (MF) on the erythrocytes of intact Wistar rats by weight 140-170 g induces the property to stimulate the immune response on sheep's T-dependent antigen-erythrocytes and on bull serum albumin in allogenic transference. The most expressed immune stimulated effect is induced by the heavy erythrocytes, which are effected by magnetic field. The warming and vibration don't induce the immunomodulating characteristics in erythrocytes. By spleen cells sticking to glass, the modified UVR and MF, the heavy erythrocytes induce factors (differentiated in mass), which stimulate the development of immune response to T-dependent antigens of sheep's erythrocytes and of bull serum albumin. These factors depress the function of antigen-specific T-suppressors and induce the immune-suppressive characteristics in light erythrocytes.     

Interaction of allogeneic lymphocytes and precursor cells during the primary and secondary immune response]

A mixture of lymph node cells from CBA mice and spleen cells from C57Bl/6J mice stimulated by the cheep erythrocytes fro the first or second time was transplanted in the lethally irradiated mice (CBA X C57Bl/6j)Fl. The interaction of allogenic cells during the secondary immune response was accompanied by the complete inactivation of antibody producents. Under the ratio of interacting cell elements 1 : 1-1 : 2, 93-96% of precursor cells and 98% of antibody forming cells were inactivated. Under the ratio 1 : 5, the index of inactivation of precursor cells fell down to 35%. During the primary response, under the ratio 1 : 1, only 20-48% of precursor cells and 68% of antibody forming cells were inactivated. Under the ratio 1 : 2, no inactivation of precursor cells was observed and, under the ratio 1 : 10, the antibody formation was stimulated. Following the delayed by 1-3 days transplantation of CBA lymphocytes, the cooperative effect was registered with respect to the spleen cells from C57Bl/6J mice stimulated by the erythrocytes for the first time. The interaction of allogenic cells resulted in the 3-4-fold increase in the number of antibody forming cells.     

Specificities of heterologous antisera against human leukaemia cells. 2. Reactions against fetal liver cells and absorption studies with fetal tissue.

Rabbit or goat antisera directed to ALL and AML cells were investigated in cytotoxicity tests with fetal liver cells as targets. After absorption with erythrocytes and spleen cells from allogenic donors the antisera killed fetal liver cells. There was no reaction with remission leukocytes or blood leukocytes from normal donors. Treatment with fetal tissue removed the activity of the AML and ALL antisera against ALL cells but not of the AML antisera against AML cells. This indicates the existence of at least two antigens on the surface of AML cells, one antigen is common with ALL cells and of fetal origin and another one seems to be characteristic of AML cells and not of fetal origin. Because treatment with fetal tissue removed all activity of the ALL antisera it can be assumed that leukaemia-associated antigens on ALL cells are of fetal origin.     

Comparative analysis of the adjuvant action of fab-fragments of normal rabbit IgG obtained using pepsin and papain]

The capacity of Fab fragments of normal rabbit IgG to enhance the immune response to sheep erythrocytes in the homologous recipients was determined by the structure of the C-terminal part of the heavy chain Fd fragment. This followed from the fact that pepsin F(a')2 and Fab' fragments enhanced considerably the hemagglutinin production and proliferation of the antibody-forming cells in the spleen, whereas papain Fab fragments, used in the same dose as the pepsin fragments, possessed only negligible adjuvant activity. As shown the adjuvant activity of pepsin and papain fragments displayed an inverse correlation with the titres and papain homoreactants in the sera of the homologous recipients. The data obtained suggested that the target cells for Fab fragments were lymphocytes carrying cytophilic homoreactants as receptors for the fragments.     

Receptors for IgG and complement in human spleen lymphoid cells. Preferential binding of particulate immune complexes through complement receptors.

Human lymphoid spleen cells attached to Petri dishes by poly-L-lysine bind 51Cr-labeled erythrocytes coated with IgG antibodies or complement but not uncoated erythrocytes or those coated with IgM antibodies. The number of erythrocytes bound through complement receptors is several times larger than that bound through IgG receptors. Increasing up to five times the number of IgG molecules on the red blood cells only leads to a slight increase of binding. However, the addition of complement to the IgG-coated erthrocytes increases 10 times the binding to spleen cells, even in the presence of an excess of normal IgG. These results can be explained by postulating that there is a larger number (or greater affinity) of spleen cell receptors for complement than that of spleen cell receptors for IgG.     

Exogenous protease promotes specific antibody forming cell response in vitro

This report presents results from experiments which evaluated the effect of exogenous protease on the in vitro antibody-forming cell (AFC) response of hamster lymphocytes to sheep erythrocytes (SRBC). In the presence of fetal calf serum, trypsin and papain, but not thermolysin, α-chymotrypsin, thrombin, and submaxillary protease, were able to enhance the quantity of AFC which developed. Prior incubation of antigen with proteases had no effect on subsequent antigenicity. The following observations were made: (1) Addition of protease to the culture system enhanced the AFC response only if added in the first 48 hr of the assay. (2) Proteases were able to enhance the development of AFC in lymph node and spleen cell cultures lacking fetal calf serum for 24 hr. (3) When papain was added to spleen cell cultures which normally produce fewer AFC than lymph node cells (LNC) it promoted the development of a 6- to 10-fold increase in AFC causing the magnitude of the response to match the AFC response expected in LNC cultures. These data support a role for a proteolytic event in lymphocyte activation by specific antigens.     

Glycophorin A interferes in the agglutination of human erythrocytes by concanavalin A. Explanation of the requirement for enzymic predigestion.

Human erythrocytes become agglutinable with concanavalin A (Con A) after treatment with various proteinases or neuraminidase. The extent of agglutinability achieved with different enzymes is, however, different: Pronase, papain, trypsin, neuraminidase and chymotrypsin enhance the agglutinability in decreasing order, the last being barely effective. The actions of the enzymes on band 3, the Con A receptor, do not correlate with their abilities to increase the agglutinability: Pronase, papain and chymotrypsin cleave the protein, but not trypsin or neuraminidase. No significant differences are found in the number of Con A-binding sites or the affinities for the lectin between the normal and trypsin- or Pronase-treated cells. Thus the receptor does not seem to play a role in determining the Con A-agglutinability of erythrocytes. On the other hand, the cleavage of glycophorins, especially glycophorin A, and the release of sialic acid (in the peptide-bound form) are well-correlated with the enhancement in agglutination after the action of proteinases. The release of sialic acid by graded neuraminidase digestion and the increase in Con A-agglutinability show a correlation coefficient of 0.88. The major inhibitory role of glycophorin A in the process is indicated by the agglutination of En(a) heterozygous erythrocytes; the cells, known to bear about 50% glycophorin A molecules in their membrane, are agglutinated approximately half as well without proteolysis as are the trypsin-treated cells. Possible mechanisms by which glycophorin A could affect Con A-mediated agglutination are discussed.     

Complement inhibitor(s) released by leukocytes. III. Evidence for a "new" C1 inhibitor in the supernatants of short-term cultures of mouse spleen and thymus cells.

Mouse spleen or thymus cells in short-term culture release a factor, designated S, that binds to sheep erythrocytes (E). Supernatant-treated sheep erythrocytes (SE) are capable of fixing and transferring the activated first component of guinea pig complement. SEC1, however is not capable of initiating hemolysis by the rest of the complement components. SE is capable of binding but not activating native C1; native C1 bound to SE seems irreversibly inhibited. Evidence is presented that S may be the same factor as the previously described inhibitor released by mouse spleen or thymus cells that inhibits the utilization of C2 by EAC14.     

Complex Effects of Papain on Function and Inhibitor Sensitivity of the Red Cell Anion Exchanger AE1 Suggest the Presence of Different Transport Subsites

Band 3 (AE1), the anion exchanger of the human erythrocyte membrane, mediates not only fluxes of small hydrophilic anions (e.g., chloride, oxalate), but also the flip-flop of long-chain amphiphilic anions (e.g., dodecylsulfate). Treatment of erythrocytes with papain, long known to inhibit the transport of the former type of anions, accelerates the transport of the latter type. In an attempt to elucidate the basis of these opposite responses to papain, several small amphiphilic arylalkyl sulfonates and -sulfates were tested for the response of their transport, via AE1, to papain. Although all these probes are most likely transported by a flux and not by flip-flop, their transport was inhibited by papain only in some cases, but accelerated in others. Different responses to papain therefore most likely do not reflect differences between transport by flux or by flip. The transports of different species of anions also differed considerably in the changes of their sensitivity, to noncovalent and some covalent inhibitors, brought about by papain treatment. While oxalate transport remained as sensitive as in native cells, transports of small amphiphilic anions lost their sensitivity to a major extent, regardless of the inhibition or acceleration of their transport by papain. The results are discussed in the light of present concepts of the structural organisation of AE1, and interpreted in terms of a model of different transport subsites for different species of anions in this transporter. Received: 20 June 2000/Revised: 1 November 2000     

Relationship of lymphotoxin secretion and DNA synthesis in the human mixed lymphocytes reaction in vitro.

Influenza virus particles, inactivated with formalin, have been covalently bound to cyanogen bromide-activated Sepharose beads (Se-vi beads). Preservation of the hemagglutination properties of the viral particles enabled a strong binding of pigeon or human group O erythrocytes (PRBC or HoRBC) to these Se-vi beads. The conditions for preparation of PRBC- or HoRBC-Se-vi columns are described.Spleen cell suspensions from mice immunized with the above erythrocytes were considerably depleted of cells forming hemolytic plaques (PFC) against the corresponding erythrocytes after passage through these columns. In the case of cells from nonimmunized mice, the depletion is still greater and reaches up to 95–100%. However, the number of PFC reactive to unrelated erythrocytes is not affected in the filtered population. Specifically attached cells recovered from the Se-vi-RBC columns passed with normal spleen cells are considerably enriched in the number of PFC against homologous erythrocytes. Syngeneic irradiated hosts transferred with filtered cells are able to give a normal primary PFC response against heterologous, but not against homologous RBC up to the 12th day after immunization. These results are discussed in relation to the problem of precommitment of specific PFC precursor cells.     

Effect of glutaraldehyde treatment on enzyme-loaded erythrocytes.

In principle, enzyme-loaded erythrocytes can be used as a vehicle for enzyme replacement therapy in lysosomal storage diseases. Glutaraldehyde treatment renders these erythrocytes more resistant to lysis without inactivating the enzymes that have been entrapped inside them. Glutaraldehyde treatment does not prevent ingestion of enzyme-loaded erythrocytes by macrophages in vitro so that these cells can be used to deliver enzymes to lysosomes. In vivo, the glutaraldehyde-treated cells are quickly removed from the circulation by the spleen or liver. The degree of glutaraldehyde treatment allows the erythrocytes to be targeted either to the spleen (low glutaraldehyde concentrations) or to the liver (higher glutaraldehyde concentrations).     

Role of membrane integral proteins in the modulation of red cell shape by albumin, dinitrophenol and the glass effect

Defatted serum albumin is found to induce a cup shape in erythrocytes. At 40 mg/ml of albumin, approx. 80% of washed erythrocytes possess this morphology, which can be reversed to disc shape by dinitrophenol. Erythrocytes treated with trypsin, papain, pronase or neuraminidase show enhanced susceptibility to cup formation by albumin; however, chymotrypsinized erythrocytes exhibit a normal response. Red cells treated with concanavalin A (but not its succinylated derivative) show resistance to the cupping effect of albumin as well as the crenating effects of dinitrophenol and glass. The resistance develops after about 20 min following the exposure of cells to the lectin, and is rapidly abrogated on removal of the bound lectin by alpha-methylmannoside. Incubation of the concanavalin A-exposed cells at low temperature leads to prolongation of the time required to achieve the resistance. These results indicate an involvement of membrane integral proteins in mediating the shape modulating effects of albumin, dinitrophenol and exposure to glass.     

Immunosuppressive activity of human seminal plasma. I. Inhibition of in vitro lymphocyte activation.

A high molecular weight fraction prepared from human seminal plasma by gel filtration chromatography suppresses human lymphocyte transformation and DNA synthesis induced by mitogens (PHA, Con A, PWM), antigens (Candida albicans, tetanus toxoid), and allogenic cells. This same fraction also suppresses the stimulated response of mouse lymphocytes to allogenic cells and to various mitogens, including T cell-dependent and T cell-independent mitogens. The induction, but not the expression, of cell-mediated cytotoxicity is also suppressed. Similar high molecular weight fractions suppress the in vitro humoral response of mouse spleen cells to both a T cell-dependent (SRBC) and a T cell-independent (DNP-F) antigen. The high m.w. fraction exhibited in vitro suppressive activity at concentrations of 0.1 to 1.0 mg/ml which corresponds to a 1/50 or greater dilution of human seminal plasma. These observations support the concept that a local immune response against sperm in the female reproductive tract is actively suppressed by a component in seminal plasma.     

Differences in the properties of specific T-suppressors and cytotoxic T-lymphocytes immune to H-2 complex antigens

Intravenous immunization of mice with a large dose of gamma-irradiated allogenic spleen cells gives rise to specific suppressor T cells and cytotoxic lymphocytes (CTL). However, being optimal form suppressor T cell induction, these conditions of immunization are not conducive to identification of CTL unless they are enriched by elution from the allogenic target monolayer. Unlike CTL, specific suppressor T cells are highly susceptible to gamma-irradiation while their precursors differ from those of CTL by high susceptibility to cyclophosphamide and hydrocortisone.     

Influence of the maternal effect on allogenic inhibition of hematopoietic stem cells]

Bone marrow cells (0,5-10(6)) of female mice of CBA or C57BL strains were injected intravenously to lethally irradiated CBA, C57BL/6, (femaleCBA X maleC57BL/6)F1 and (femaleC57BL/6 X maleCBA)F1 mice. Spleen of recipients as assayed for colony count on the 9th day after bone marrow transplantation by the method of Till and McCullouch. Stem cells of CBA mice demonstrated failure of allogenic inhibition in (CBA X C57BL/6)F1 hybrid mice and formed the same number of colonies as in the spleen of syngenic recipients. The level of allogenic inhibition of CBA stem cells transplanted to (C57BL/6 X X CBA)F1 hybrid mice was 50%. Bone marrow cells of C57BL/6 mice formed colonies in spleen of (CBA X C57BL/6)F1 mice at least in 20 times less than in syngenic combination. In the transplantation of bone marrow from C57BL/6 mice to (C57BL/6 X CBA)F1 hybrid mice the allogenic inhibition was less pronounced (77-85%) as compared with the transfer of cells to (CBA X C57BL/6)F1 hybrid mice (95%). The sex of a recipient did not influence the number of formed colonies. The different level of allogenic inhibition of parental stem cells can not be explained by the effect of linkage with sex as the female of reciprocal hybrid mice have identical structure of sex chromosomes (X(CBA)XC57BL/6). The data obtained indicate that the maternal effect affects allogenic inhibition of stem cells in parent--F1 system. It is possible that the maternal influence may be determined by cytoplasmic factors of inheritance which affect the expressivity of recessive genes Hh, controlling the inheritance of specific haematopoietic cell antigens.     

A B Lymphocyte Mitogen Extracted from a Fungus Peziza vesiculosa1

A water-soluble mitogen was extracted with hot-water from the fruiting bodies of a fungus, Peziza vesiculosa, collected in the wild. The active substance, named vesiculogen, was able to stimulate selectively murine B cells because mitogenic activity was observed in the spleen cell cultures of congenitally athymic nude mice, but not in the thymus cell cultures. The possibility that the mitogenicity of vesiculogen was due to lipopolysaccharide was denied completely by the following evidence: 1) lipopolysaccharide in vesiculogen was undetectable (less than 0.001% in the Limulus test), 2) vesiculogen was able to stimulate strongly DNA synthesis of spleen cells from C3H/HeJ mice, and 3) the mitogenic activity of vesiculogen was not inhibited by polymyxin B. Vesiculogen increased antigen-nonspecifically the number of direct plaque forming cells to sheep erythrocytes, horse erythrocytes, and trinitrophenylated-horse erythrocytes. This result shows that vesiculogen acts as a polyclonal B cell activator on murine spleen cells.     

Enhanced antibody response in retrovirus-infected mice treated with endotoxin or nontoxic polysaccharide derivative

Friend leukemia virus (FLV) is a retrovirus which causes marked suppression of the immune response of genetically susceptible mice. In the present study the depressed antibody response to sheep erythrocytes by spleen cells from FLV-infected mice was partially reversed by injection of either a bacterial endotoxin or a nontoxic polysaccharide derivative directly into infected mice or by addition to spleen cell cultures from these mice immunized in vitro with sheep red blood cells (SRBC). The endotoxin and PS in a dose-related manner markedly increased the antibody responsiveness of the spleen cells to SRBC. Thus these results indicate that the nontoxic polysaccharide derivative has properties equivalent to the toxic endotoxin in enhancing the antibody responsiveness of FLV-suppressed spleen cells to a T-cell-dependent antigen like SRBC.     

Characteristics of the immunosuppressive activity of lymphoid organ cells in the process of sensitization with ram erythrocytes and exposure to antilymphocyte serum

In the adoptive transfer of cells obtained from the thymus, lymph nodes and the spleen to intact syngeneic animals the suppression of immune response was induced by lymph node cells. If the donors were previously sensitized, the cells of the thymus and lymph nodes showed suppressive activity in the adoptive transfer test. A single injection of antilymphocytic serum to the donors of lymphoid cells, previously sensitized with sheep red blood cells, enhanced the immunosuppressing action of thymocytes and lymph node cells.     

Immunomodulating activity of polyunsaturated phospholipids and regulators of energy metabolism in toxic anemia]

Possible potentiation of the immunomodulating effects of carbohydrate and lipid metabolism regulators by their use in combination with polyunsaturated phospholipids was studied. The polyunsaturated phospholipids in toxic anemia icreased the immunomodulating effects of thiamine and inosine which activated glucose catabolism in erythrocytes. The combined use of the polyunsaturated phospholipids and thiamine normalized the oxidation--energy status and lowered manifestation of the immunosuppressing properties of light erythrocytes in laboratory rats exposed to hemolytic poison. The use of the combination of the polyunsaturated phospholipids and inosine normalized the oxidation--energy status and induced manifestation of the immunomodulating properties in heavy erythrocytes of the poisoned rats. The globulin fraction of the rat serum containing antibodies to erythrocytes of the poisoned rats exposed to the polyunsaturated phospholipids and inosine increased the immunity status of the poisoned rats treated with the above mentioned agents. Carnitine and biotin in combination with the polyunsatured phospholipids showed no effect on the phagocytic and metabolic activity of leukocytes and the immunity status of the rats exposed to hemolytic poison.