Altered humoral immune response to sheep red blood cells by sheep erythrocyte-soluble hemolysate.

Adult C3H/He mice were rendered unresponsive to a primary injection of sheep red blood cells (SRBC) by pretreatment with sheep hemolysate supernatant (SHS) or subfractions of SHS isolated by column chromatography. The following effects of SHS on the immune response were observed: SHS did not kill antigen-reactive cells, it did not prevent the release of antibody by cells actively synthesizing and secreting antibody, and SHS-induced tolerance was not inhibited or abrogated by methods which terminate or abolish tolerance. In addition, cell-mediated responses were not affected in animals whose humoral responses were suppressed; however, the secondary plaque-forming cell (PFC) response was enhanced by SHS treatment. SDS gel electrophoresis revealed SHS to contain several proteins ranging from 12,000 to approximately 500,000 daltons.     

Naturally abundant basophils in the snapping turtle, Chelydra serpentina, possess cytophilic surface antibody with reaginic function

Basophils constitute 50 to 63% of the blood leukocytes in Chelydra serpentina, the snapping turtle. Immunoglobulin (Ig) on the surface of the turtle basophil was detected by indirect immunofluorescence by using an IgG fraction from rabbit anti-turtle Ig serum (RATIg) and a fluoresceinated goat anti-rabbit antibody incubated at 4 degrees C. However, when the cells were incubated with RATIg at 22 degrees C, the basophil number, as determined by Wright's stain and neutral red counts, decreased dramatically. This morphologic evidence of degranulation was directly proportional to the antiserum concentration. Degranulation also correlated with cell histamine release (r = 0.73). In other experiments, turtle basophils were found to express antigen-specific surface Ig after immunization with sheep red blood cells (SRBC). Washed basophils from immunized turtles formed basophil-SRBC rosettes in vitro. Basophils from control turtles did not. Basophil-SRBC rosettes could also be induced by in vitro passive sensitization by preincubation of normal turtle basophils in the SRBC immune turtle sera. This study shows clearly that the turtle basophil has an immune capacity analogous to the mammalian basophil/mast cell. This study also contains the first direct evidence for the existence of reaginic antibody (or antibodies) in an ectothermic vertebrate. Finally, C. serpentina is proposed as a unique animal model for the study of basophil function.     

Effect of mitogens on suppression of antibody formation and proliferation in dense cultures

A study was made of the effect of mitogens on general proliferation and primary immune response to sheep red blood cells in density-inhibited cultures of mouse spleen cells. The mitogens applied included fetal calf serum and both B cell- and T cell-specific mitogens (dextran sulfate, LPS and ConA). Experiments with 3H-thymidine incorporation demonstrated that the proliferation was equally enhanced by any mitogen in both optimal and density-inhibited cultures. The mitogens did not remove the density inhibition of antibody formation.     

Time and dosage dependence of immunoenhancement by murine type II interferon preparations.

Preparations of Type II immune induced Interferon enhanced the plaque forming cell response of mice to sheep red blood cells both in vivo and in vitro. The enhancement of the antibody response was dependent on the dosage of interferon used and the time of administration of interferon. The expression of the antiviral and immuno-enhancing activities of Type II interferon preparations shared several physical-chemical properties, including pH 2 lability and heat stability. The plaque forming cell response to lipopolysaccharide, a T-independent antigen, could not be enhanced by treatment with Type II interferon. In addition, treatment of spleen cell cultures of nude thymic deficient mice with Type II interferon could not cause an enhancement of the plaque forming cell response to lipopolysaccharide. These data suggest that the immunoenhancing effect of Type II interferon on antibody responses is produced by an effect on T lymphocytes in contrast with the immunosuppressive effect which appears to be mediated through an effect on B lymphocytes.     

Role of mediators of cellular hypersensitivity in the stimulation of the antibody formation to unrelated antigen

The effect of a concurrent delayed hypersensitivity reaction on the antibody response to sheep red cells was assessed by a plaque assay. Guinea pigs with delayed hypersensitivity to tuberculin purified protein derivative (PPD) or egg albumin showed an increased antibody response to sheep red cells when the cells were injected intravenously at the same time as PPD or egg albumin. This effect was transferred to normal guinea pigs by serum from guinea pigs with delayed hypersensitivity to PPD or egg albumin taken 24 hr after injecting the corresponding antigen. Supernatants containing migratory inhibitory factor were prepared by incubating lymphocytes from sensitized rabbits with antigen. These supernatants were injected with sheep red cells and gave rise to an enhanced plaque response. Similar results were obtained with supernatants from normal rabbit thymus cells. The role of mediators of delayed hypersensitivity in enhancing antibody formation and in T cell/B cell cooperation is discussed.     

Plasmodium yoelii: antibody response in resistant and susceptible mouse strains

The numbers of antigen-reactive antibody-secreting cells, levels of parasite antigen-specific serum antibodies and numbers of red blood cells staining positive for surface immunoglobulin were determined for susceptible and resistant mouse strains following infection with Plasmodium yoelii 17x. As a control, these parameters also were measured using antigen prepared from normal red blood cells. The relatively susceptible C57BL/6 mice produced more antigen-specific antibody-secreting cells and had higher levels of immunoglobulin positive red blood cells than did DBA/2 mice, but the DBA/2 mice had more antigen-specific IgG in their sera. Both mouse strains possessed cells secreting antibody reactive with soluble normal red blood cell antigen; however, C57BL/6 mice had more IgG positive unparasitized RBC than did DBA/2 mice. Despite possessing fewer antibody positive normal RBC, DBA/2 mice had significantly higher levels of serum antibodies that reacted with soluble red blood cell antigen. These data indicate that levels of serum antibody may not reflect the amounts of antibody produced and that use of any single assay to assess the magnitude of the antibody response may give rise to misleading results.     

Lymphocyte subsets involved in tumor-activated nonspecific feedback suppression

Cells from the spleen, lymph nodes, and peritoneum of DBA/2 mice bearing a subcutaneous tumor mediate nonspecific suppression of an in vitro antibody response to sheep red blood cells (SRBC) when cocultured with a normal T-cell subset(s). The spleen cells from the tumor-bearing mouse required for the suppression bear the Lyt 1 and Ala 1 surface markers characteristic of "inducer" T cells and activated cells, respectively. The activity of this cell population is also sensitive to irradiation. The normal T-cell subset which cooperates in the suppression bears the Qa-1 surface antigen which has been associated with suppressor cell precursors in several systems but lacks detectable surface Lyt 1 and 2 markers. Suppression of antibody responses in spleen cell cultures from tumor-bearing mice alone could also be elicited, but only when increased numbers of cells were cultured. These data are consistent with the theory that a tumor-activated, Lyt 1+ T-cell subset has the capacity to nonspecifically suppress immune responses by activating a Qa-1+ subset(s) of T suppressor cells, perhaps via feedback signals.     

Immunomodulating activities of staphylococcal enterotoxins. I. Effects on in vivo antibody responses and contact sensitivity reaction

The immunomodulating effects of staphylococcal enterotoxins on in vivo immune responses in C57BL/6 mice were examined. Of the five serological types A (SEA), B, C, D, and E (SEE), only SEA and SEE markedly suppressed the antibody response to sheep red blood cells (SRBC) when injected 1 day before or on the day of immunization with SRBC. Further study of SEA revealed that it did not affect the antibody response to a thymus-independent antigen, salmonella flagella, but did affect the T-cell-mediated immune response. Contact sensitivity to dinitrofluorobenzene (DNFB) was suppressed when SEA was injected before sensitization or before challenge with DNFB, indicating that SEA affected both the afferent and efferent phases of DNFB contact sensitivity. As the suppression of DNFB contact sensitivity could be transferred by anti-Thy-1.2 antibody-sensitive spleen cells of SEA injected donors into normal or DNFB-sensitized recipients, the suppression was thought to be an active one. However, SEA could augment the DNFB contact sensitivity when injected on the third day after sensitization with DNFB. These results indicate that the immunomodulating effects of SEA can be mediated by the T-cell function.     

Experimental Mycoplasma pulmonis infection of rats suppresses humoral but not cellular immune response

Humoral antibody response to sheep red blood cells and cellular immune response to bovine serum albumin were studied in Mycoplasma pulmonis infected, adult, male Sprague-Dawley rats. The hemagglutinating antibody response to sheep red blood cells was evaluated at 0, 3, 5, 7, 14, 21 and 28 days postinfection. Antibody titers during all days postinfection were depressed significantly (p less than 0.05) in infected rats as compared to noninfected controls. Cellular immune responses were evaluated by a delayed hypersensitivity response. Rats were sensitized at 0, 3, 5, 7, 14, 21 or 28 days postinfection with bovine serum albumin and challenged with heat aggregated bovine serum albumin 7 days later. Cell-mediated immune responses in infected rats were not significantly different at any point from controls. These results indicate that M. pulmonis infection in rats suppresses the humoral antibody response to sheep red blood cells, but not the cellular immune response to bovine serum albumin.     

Distinctive functional properties of human blood L lymphocytes: a comparison with T lymphocytes, B lymphocytes, and monocytes.

Human blood lymphocytes with high affinity Fc receptors have been operationally named L lymphocytes because of membrane-labile IgG markers. L lymphocytes lack membrane-incorporated immunoglobulin and do not form rosettes with sheep red blood cells coated with IgM antibody and mouse complement. These lymphocytes are capable of binding IgG in normal human serum at 4 degrees C and will form rosettes with human lymphocytes coated with Ripley IgG. In this study, functional in vitro properties of isolated L lymphocytes were compared with T lymphocytes, B lymphocytes, and monocytes. To obtain these mononuclear populations, first, plastic adherent monocytes were harvested. T lymphocytes were then isolated by centrifugation of E rosette-forming cells, and other rosetting techniques were employed to isolate L and B lymphocytes by negative selection. The functional properties of L lumphocytes were completely unlike those of T cells, B cells, or monocytes. L lymphocytes did not proliferate in response to mitogens, soluble antigens, or cell surface antigens. Moreover, this population could not replace monocytes in helping T lymphocytes respond to concanavalin A and pokeweed mitogen. Once T cells were supplemented with monocytes, however, the addition of L lymphocytes to the culture greatly enhanced the T lymphocytes proliferative response to phytohemagglutinin, concanavalinA, purified protein derivative (PPD), and streptokinase/streptodornase. L lymphocytes were not a subset of B cells. They did not spontaneously develop surface Ig in culture, and pokeweek mitogen could not induce them to transform and generate cytoplasmic Ig detectable by immunofluorescence. Mixtures of B cells and T cells responded to pokeweed mitogen better than do T cells alone. In contrast, enhanced reactivity with L and T cell combinations was not observed. Another sharp difference between these two populations was the stimulator capacity of each in mixed lymphocyte culture. When B and L lymphocytes were carefully monocyte-depleted, only B cells were effective stimulators of autologous and allogeneic lymphocytes. In comparison with T cells, B cells, and monocytes, L lymphocytes were the only effective killers of human blood lymphocytes sensitized with IgG. L lymphocytes, then, have cytotoxic potential, but cannot proliferate in response to various stimulants or become antibody-producing cells. These findings suggest that L lymphocytes comprise a third lymphocyte population.     

Immunologic characterization of hairy cell leukemias in continuous culture.

The immunobiologic characteristics of three continuous cell lines established from hairy cell leukemia cells were investigated. All three cell lines continued to produce tartrate-resistant acid phosphatase, the enzymatic marker of hairy cells. Two of these cell lines were B lymphoid in nature. They carried Fc and C receptors, had surface and internal immunoglobulin, and did not form spontaneous sheep red blood cell rosettes. Experiments employing biosynthetic radiolabeling of immunoglobulin demonstrated distinctive immunoglobulin kinetics for each of these two hairy cell lines. One cell line remained quite similar to the original hairy cells from which it was derived whereas the other B lymphoid hairy cell line had undergone a switch in the immunoglobulin isotype produced. The third hairy cell leukemia line was shown to be of thymic derivation. These cells formed spontaneous sheep red blood cell rosettes and did not carry Fc or C receptors. The spontaneous sheep red blood cell rosette-forming cells contained tartrate-resistant acid phosphatase. They did not possess surface on internal immunoglobulin and did not synthesize immunoglobulin in vitro. Hairy cell leukemia cells maintained in permanent cell culture retain their immunobiologic properties and offer the opportunity for indepth study of these unusual cells.     

Tumor-mediated immunosuppression: Prevention by inhibitors of prostaglandin synthesis

The addition of MC16 tumor cells (a prostaglandin E₂-producing cell line induced in C57B1/6J mice by methylcholanthrene) to cultures of normal syngeneic spleen cells inhibits the antibody response of these cells to sheep red blood cells. This inhibition can be blocked by adding to the cultures prostaglandin synthetase inhibitors, such as indomethacin, flufenamic acid and aspirin. These MC16 tumor cells are also immunosuppressive . Mice bearing the syngeneic MC16 tumor become unresponsive to sheep red blood cells as the tumor grows. As in the test system, inhibitors of prostaglandin synthetases seem to block the immunosuppressive activity of MC16 cells since tumor-bearing mice, treated therapeutically with indomethacin, responded normally in their production of antibody to sheep red blood cells.     

Enhancement of antibody production by lysophosphatidylcholine and alkylglycerol

Inflammation products of normal and cancerous tissues, lysophosphatidylcholine and dodecylglycerol, were tested for their adjuvant effect on the antibody response. Mice treated with these agents and immunized with sheep erythrocytes simultaneously or at 3 days posttreatment developed a greatly enhanced antibody production as demonstrated by the Jerne plaque assay. Mice immunized at 3 days postadministration of agents did not significantly produce enhanced antibody-secreting cells as compared with those of mice simultaneously immunized. Since the mechanism of macrophage activation by lysophospholipids requires contribution of B and T cells, BALB/c-nu/nu mice treated with these agents and subsequently immunized with sheep erythrocytes did not produce antibodies. However, conditioned medium of in vitro-treated BALB/c-nu/nu B cells efficiently transmitted a signal to untreated BALB/c +/+ T cells for enhanced macrophage ingestion activity. This observation suggests that lysophospholipid-activated macrophages and T cells efficiently transmitted antigenic signal to the antibody-producing B cell population. Therefore, we conclude that these lipid metabolites have dual beneficial effects for the host by enhancing phagocytosis and antibody production. Thus, lysophosphatidylcholine and dodecylglycerol have potential practical application as adjuvants that could be administered separately or in combination with antigens.     

Lipopolysaccharide-induced suppression of the primary immune response to a thymus-dependent antigen.

The immune response to a thymus-dependent antigen was depressed in vivo and in vitro in spleen cells from mice injected with LPS i.p. a few days before challenge with the antigen. Spleen cells from LPS-injected mice could, however, respond with increase DNA synthesis after activation with polyclonal B and T cell activators in vitro. The LPS-activated spleen cells could actively suppress normal cells in their response to the antigen sheep red blood cells. The suppressor cells contained in the LPS-activated spleens were most likely B lymphocytes, and the possible mechanism for their inhibitory function is discussed.     

Influence of human thymocytes on B-lymphocyte differentiation in man.

Human thymocytes from children less than 6 years of age were tested for their influence on differentiation of normal B cells. The addition of either thymocytes or a culture supernatant from thymocytes to normal peripheral blood lymphocytes (PBL) enhanced pokeweed mitogen-induced B-cell differentiation as tested in a plaque-forming assay for antibody to sheep red blood cells. The thymocytes, however, could not substitute for T lymphocytes in cultures of PBL which had been previously depleted of T lymphocytes. Further, prior treatment of thymocytes with concanavalin A did not result in generation of suppressor cells for either B-cell differentiation or for the responses of PBL to mitogens. Thus, although thymocytes were functionally immature by these assays as compared to mature T lymphocytes they exerted an influence on B-cell differentiation in cultures of normal peripheral blood lymphocytes.     

Effect of an orally applied herbal immunomodulator on cytokine induction and antibody response in normal and immunosuppressed mice

The influence of the oral administration of a herbal immunomodulator, consisting of an aqueous-ethanolic extract of the mixed herbal drugs Thujae summitates, Baptisiae tinctoriae radix, Echinaceae purpureae radix and Echinaceae pallidae radix, on cytokine induction and antibody response against sheep red blood cells was investigated in mice. The treatment of the animals with the extract caused no enhancement of the cytokine titers in the serum. Spleen cells isolated from the treated mice, however, produced higher amounts of IL-2, IFNgamma and GM-CSF ex vivo in comparison to spleen cells isolated from control animals, especially after additional stimulation by lipopolysaccharides or concanavalin A. The application of the extract also triggered the production of IL-1 and TNFalpha by peritoneal macrophages ex vivo. The influence of the herbal extract on the antibody response was examined by the plaque forming cell assay. The administration of the extract caused a significant enhancement of the antibody response against sheep red blood cells, inducing an increase in the numbers of splenic plaque forming cells and the titers of specific antibodies in the sera of the treated animals. In mice, immunosuppressed by old age or additional treatment with hydrocortisone, the therapy with the extract resulted in a normalization of the antibody response against sheep red blood cells.     

ELECTROKINETIC PHENOMENA. III : THE "ISOELECTRIC POINT" OF NORMAL AND SENSITIZED MAMMALIAN ERYTHROCYTES

A survey of the published electrophoretic mobilities of certain mammalian red cells reveals that the isoelectric points accorded to these cells are the result of equilibria incidental to red cell destruction. The electrophoretic mobilities of normal washed sheep and human cells have now been studied in 0.85 per cent NaCl solutions from about pH 3.6 to 7.4. All measurements were made within 2 minutes of the preparation of the suspension of red cells. In no case was reversal of sign of charge observed under these conditions. Reversal of sign of charge occurred only after sufficient time had elapsed to permit sufficient adsorption of the products of red cell destruction. There is little change in mobility as the pH of the medium is decreased. Reversal of sign of charge does occur in the presence of normal and immune (anti-sheep) rabbit sera. The isoelectric point determined under these conditions does not appear to be connected specifically with the immune body but is perhaps associated with phenomena incidental to red cell destruction and the presence of serum. The characteristic lowering of mobility by amboceptor occurs, however, from pH 4.0 to pH 7.4. The curves of mobility plotted against pH for normal and for immune sera support the viewpoint that the identity of the isoelectric points for normal and sensitized sheep cells is not primarily concerned with the immune reaction. It is most unlikely that an "albumin" or a "globulin" surface covers red cells with a complete protein film. Although serum protein reacts with red cells in acid solutions, this is not demonstrable for gelatin. The lowering of mobility usually ascribed to anti-sheep rabbit serum may also occur, but to a lesser degree, in normal rabbit serum. This diminution of mobility is not, in the first place, associated with sensitization to hemolysis induced by complement. This supports the view that only a very small part of the red cell surface need be changed in order to obtain complete hemolysis in the presence of complement.     

Cellular immunity in chronic Theiler's virus central nervous system infection.

After (IC) inoculation of the DA strain of TMEV, SJL/J mice develop chronic CNS infection with marked mononuclear cell infiltration of spinal cord leptomeninges and white matter and concomitant demyelination. In the present study the temporal course of cell-mediated and humoral immune responses to virus were measured in this infection. It was shown that chronic TMEV infection is associated with the development of immunologically specific spleen cell reactivity as judged by in vitro incorporation of 3H-TdR into DNA in response to inactivated TMEV antigen. Spleen cell reactivity is first detectable about 2 months after infection, persists for at least 1 year, and correlates with the temporal development of serum-neutralizing antibody. The late development of sensitized spleen cells is not the result of an immunosuppressive effect of this virus infection since infected mice exhibit normal spleen cell proliferative responses to T cell mitogens and produce normal antibody responses to a heterologous protein antigen, sheep red blood cells. In addition, anti-viral antibody inhibits virus-induced spleen cell reactivity. Finally, the antigen-reactive lymphocyte subpopulation within the spleen responsible for proliferation to TMEV antigen are T cells and not B cells.     

The role of immunity on the development of dermatitis in NC mice

In order to clarify the role of immunity on the development of dermatitis in NC mice, the following experiments were carried out. In neonatal thymectomized NC, thymic reconstituted NC-nu/nu, and passively serum transferred NC-nu/nu mice, incidence of the dermatitis was examined. Immune response to sheep red blood cells (SRBC) and number of Thy-1 positive cells in mesenteric lymph node were used as indicators of the cell mediated immunity. Although antibody to SRBC and the number of Thy-1 positive cells in neonatal thymectomized NC mice were greatly reduced, development of the dermatitis in these mice was not suppressed at all. On the contrary, thymic reconstituted NC-nu/nu mice which recovered immune response to SRBC and number of Thy-1 positive cells to the normal levels did not develop the dermatitis. Passive transfer of the serum obtained from NC mice which developed severe dermatitis, could not induce the dermatitis in NC-nu/nu mice. These results suggest that the dermatitis in NC mice is not mediated by immune mechanisms but by other complexed factors. The absence of the dermatitis in NC-nu/nu mice may be due to nu gene effects other than those of immune defect.     

The effect of the antigen-induced splenic suppressor factor on the immune response to different antigens

Quick-frozen spleen of mice immunized with sheep red blood cells was homogenized and centrifuged. Supernatant was used as a source of suppressor factor (SF). It was shown that SF inhibited antibody immune response to thymus-dependent antigens and delayed hypersensitivity reaction. SF did not inhibit antibody formation to thymus-independent antigen. SF activity disappeared after its treatment with anti-Ig immunosorbent.