Carrageenan-mediated suppression or augmentation of mitogen-induced lymphocyte proliferation

The effects of carrageenan on mitogen-induced lymphocyte proliferation were studied in the guinea pig. According to the dose used, carrageenan displayed opposite effects on thymidine uptake by spleen cells or peripheral blood leukocytes stimulated by Concanavalin A or phytohemagglutinin (25 micrograms ml-1 carrageenan increased whereas 0.25 microgram ml-1 depressed thymidine incorporation). Carrageenan, at the high concentration which increased thymidine uptake by mitogen-stimulated spleen cells, potentiated the enhancing activity of macrophages that was observed with cell suspensions containing 20% macrophages. Conversely, low concentrations of carrageenan abolished the enhancing effect of macrophages. These effects of carrageenan on lymphocyte proliferation could be explained by its activities on a macrophage functional subset rather than on the whole macrophage population.     

Effects of carrageenan-a macrophage toxic agent-on antibody synthesis and on delayed hypersensitivity in the guinea pig

Delayed hypersensitivity reactions to the carrier and antibody mediated reactions to the carrier and to the hapten have been studied in guinea pigs treated with carrageenan, a macrophage toxic agent, before or after immunization with a hapten-carrier complex. The results show that carrageenan which acted at the afferent limb of immunity neither depressed nor enhanced delayed hypersensitivity reactions but could affect antibody formation. Macrophage functions in antibody synthesis appeared to be more sensitive to carrageenan than those involved in the induction of cell-mediated immunity. Carrageenan could provide a useful tool for studying the functional heterogeneity of macrophages during the induction of the immune response.     

Immune activation by T-independent antigens: lack of effect of macrophage depletion on the immune response to TNP-LPS, PVP and dextran.

Carrageenan, a sulphated polysaccharide, and rabbit anti-mouse macrophage serum, were used to inhibit macrophage function in BALB/c mice as well as to deplete macrophages from spleen cell cultures in an attempt to determine the requirement for macrophages in the immune response to several thymus-independent antigens. Carrageenan inhibited macrophage function and was cytotoxic at low concentrations. The ability of T and B lymphocytes to undergo mitogen-induced proliferation in the presence of PHA and PLS, respectively, was not affected by in vitro exposure of lymphoid cells to carrageenan. BALB/c mice injected with carrageenan demonstrated a suppressed immune response to SRBC, a thymus-dependent antigen, but not to E. coli LPS, polyvinyl-pyrrolidone or dextran B-1355S, all of which are known to be thymus independent antigens. The sensitivity of the in vivo immune response to SRBC after depletion of macrophages by carrageenan treatment was confirmed in vitro using the Marbrook--Diener culture system. The in vitro immune response to TNP-LPS was unaffected by either carrageenan treatment or treatment of BALB/c spleen cells with AMS and complement. The results of experiments which utilized the two anti-macrophage reagents, carrageenan and AMS, both in vivo and in vitro systems, suggest that the immune response to thymus-independent antigens does not require the participation of macrophages.     

Carrageenan-Induced Suppression of T-Lymphocyte Proliferation in the Rat: Abrogation of Suppressor Factor Production by the Prostaglandin Synthesis Inhibitors,Indomethacin and ETYA

Carrageenan, an algal polygalactan reputed to be selectively toxic for macrophages, is widely employed as a tool to dissect pathways of cell-mediated immunity. In the present study, corn oil-elicited rat peritoneal macrophages after 72 h culture with 10 μg/ml Seakem® 9 Carrageenan secreted a soluble suppressor factor capable of abrogating T-cell activation by phytohemagglutinin-P (PHA). Addition of the prostaglandin synthesis inhibitors Indometha-cin or 5,8,11,14-eicosatetraynoic acid (ETYA) prevented inhibitor synthesis by Carrageenan-conditioned macrophages. Seakem ® 9 and lambda Carrageenans added directly into spleen cell cultures failed to diminish lymphocyte proliferation, but rather stimulated spleen cell division. Macrophages cultured with low concentrations of Carrageenan appeared to be activated on the basis of enhanced tumoristatic capacity against Schmidt-Ruppin sarcoma cells. Thus, macrophages activated by low concentrations of Carrageenan in vitro appear to secrete a product of arachidonic acid metabolism which is a potent inhibitor of PHA-induced spleen cell mitogenesis.     

Interleukin-2 reverses deficient cell-mediated immune responses in rheumatoid arthritis

The depressed cell-mediated immunity in rheumatoid arthritis was investigated in vivo by cutaneous hypersensitivity responses to seven antigens including tuberculin PPD, and in vitro by lymphocyte transformation to the latter antigen. In vivo 40% of rheumatoid patients were anergic compared to 2% of controls (P less than 0.001) with an associated reduction in sum score (5.9 +/- 6.5 vs 15.3 +/- 8.7, P less than 0.001). In vitro lymphocyte proliferation to PPD was also significantly depressed (P less than 0.001) and could not be reversed by indomethacin. A significant correlation between the in vivo sum scored (induration in mm) and in vitro thymidine incorporation (d/min) (r = 0.59, P less than 0.001) was found. In an attempt to overcome the depressed in vitro response the addition of a crude supernatant from a mixed lymphocyte reaction was found to return the PPD stimulated lymphocyte proliferation to the normal range. This effect was mimicked by purified IL-2 but not purified IL-1. The implications of this finding are are discussed.     

Acetylspiramycin and the immune system--II. Effects on lymphocyte proliferation, lymphokine production, delayed-type hypersensitivity and antibody production

The effects of the antibiotic acetylspiramycin (ASPM) on lymphocyte function were studied in vitro and in vivo. When added to lymphocyte cultures in vitro, ASPM inhibited splenic lymphocyte transformation induced by phytohemagglutinin (PHA), lipopolysaccharide (LPS) and antigen. It also depressed production by spleen cells of the lymphokine inducing procoagulant activity in mouse macrophages. Spleen cells from mice given ASPM orally showed enhanced responses to PHA, but normal responses to LPS. The capacity to produce lymphokine was increased early after oral ASPM and slightly decreased after prolonged administration. Oral ASPM had no effect on the production of antibodies and a very slight enhancing effect on the development of delayed-type hypersensitivity to sheep red blood cells.     

Effects of Calmodulin Antagonists Cd2+ and Trifluoperazine on Mixed Lymphocyte Culture Cell-Mediated Lysis and Antibody-Dependent Cell Cytolysis

The effects of two substances with calmodulin-antagonistic properties, cadmium and trifluoperazine, were studied on mixed lymphocyte culture-cell mediated lysis (MLC-CML) and antibody-dependent cell cytolysis (ADCC). Both cadmium and trifluoperazine readily inhibited MLC-CMC, while ADCC was inhibited only to a small extent or not at all. Trifluoperazine almost completely inhibited phagocytosis in cells whose ability to lyse antibody-coated red blood cells was unimpaired. The results suggests a striking difference in the extent in which calmodulin mediated processes take part in MLC-CML on the one hand and ADCC on the other. This indicates a profound dissimilarity in the mechanisms of these two types of cell-mediated lysis.     

Different effects of influenza virus, respiratory syncytial virus, and Sendai virus on human lymphocytes and macrophages.

Influenza virus, respiratory syncytial virus, and Sendai virus depress human cell-mediated immune responses, such as mitogen-induced lymphocyte transformation, but differ in their ability to induce other immune defense mechanisms, such as interferon production. Exposure to the different viruses resulted in depressed transformation responses to the mitogen phytohemagglutinin by affecting the function of lymphocytes, or macrophages, or both cell types.     

Cellular immunity in renal failure: depression of lymphocyte transformation by uraemia and methylprednisolone. Intra-individual consistency of lymphocyte responses to the in vitro suppressive effect of steroid

In vitro parameters of the cell-mediated immunity were assessed in patients with chronic renal failure on haemodialysis (HD). The in vitro mitogen responses of uraemic lymphocytes are depressed compared to control lymphocyte cultures. Prolonged or shortened incubation of uraemic cultures does not normalize the mitogen responses, and the difference is reflected in both DNA, RNA and protein synthesis of lymphocytes. Lymphocyte responses of control cultures incubated with uraemic plasma are similar to those of cultures with saline, and significantly stronger (p less than 0.05) than those of the uraemic lymphocyte cultures. The relative in vitro immunosuppressive effect of steroid is stronger in uraemic cultures. Thus the depressed uraemic lymphocyte responses may be associated with steroid-sensitive cellular interactions independent of incubation periods. However, both control and uraemic lymphocyte cultures have a reproducible individual in vitro lymphocyte response to the immunosuppressive effect of steroid.     

Inhibition of natural killer-cell mediated cytolysis with monoclonal antibodies to restricted and non-restricted epitopes of the leucocyte common antigen.

Three monoclonal antibodies recognizing different epitopes of the leucocyte common molecule, CMRF-11 (against the restricted or B-220 leucocyte common molecule), CMRF-12 and CMRF-26 [each against a different epitope on the non-restricted or T200 leucocyte common (CD45) molecule], were tested for their effects on lymphocyte cytotoxicity. The individual monoclonal antibodies inhibited human natural killer cell-mediated cytolysis (NK-CMC) weakly, but a mixture of CMRF-11 + 12 + 26 antibodies inhibited cytolysis more consistently and to a greater extent. This mixture did not inhibit cytotoxic T lymphocytes derived from secondary mixed lymphocyte cultures. The CMRF-11 + 12 + 26 mix was shown to inhibit a post-conjugate formation stage of lysis at the effector cell level. Inhibition of NK-CMC of a wide range of target cells, including the T-cell lines Jurkat, HSB2 and Molt 4, was demonstrated.     

Specific inhibition of allogeneic reactions with disodium cromoglycate

Milligram amounts of disodium cromoglycate (DSCG) inhibit allogeneic responses in mixed lymphocyte culture (MLC) reactions, but do not affect cell viability or suppress lymphocyte responses to either phytohemagglutinin (PHA) or pokeweed (PKW) mitogens. Preincubation of lymphocytes with DSCG is without effect, indicating that membrane binding is an unlikely explanation for inhibition. The HLA-DR tissue typing of cells in the presence of optimal MLC-inhibitory doses of DSCG is normal suggesting that MLC-reactive lymphocytes are not denied recognition of these antigens. Timed studies demonstrate that DSCG must be present continuously during the induction period, for removal of DSCG after 16 hr culture restores MLC reactivity and addition of the drug after 48 hr is without effect. Both natural killing (NK) and cell mediated lympholysis (CML) assays proceed normally in the presence of optimal MLC-inhibitory concentrations of DSCG; however, CML reactions are eliminated by the addition of drug during cytotoxic T cell priming. Background CML reactivity also disappears when lymphocytes are continuously cocultured in DSCG, implying that such killing cannot be attributed to NK activity. DSCG is said to inhibit allergic reactions by impeding calcium flux across mast cell membranes, thereby preventing degranulation, but other mechanisms are required to explain the selective effects on in vitro lymphocyte reactivity.     

Augmentation of cell-mediated responses in vitro by a monoclonal anti-helper factor antibody

Previous studies have shown that monoclonal antibody AF3.44.4 has specificity for a constant region determinant on mouse antigen-specific helper factors and that it also binds to cultured T cells with functional helper cell characteristics. The antibody synergizes with antigen to enhance in vitro antibody responses; here we demonstrate that it will also enhance cell-mediated responses in vitro such as in the generation of proliferating cells in mixed lymphocyte responses and in the generation of specific killer cells in cytotoxic T lymphocyte cultures. The mechanism of AF3.44.4-generated enhancement was investigated. Increased levels of the lymphokines IL-2 and BCDF were detected in supernatants of AF3.44.4-treated cultures but the antibody itself could not replace interleukin-2 (IL-2), and would not stimulate primed cells in the absence of antigen. This type of monoclonal antibody which augments immunological responses in an antigen-dependent fashion may provide a new class of immunostimulant and a new approach to augmenting the responses of weak immunogens.     

Strain dependence of rat macrophage natural tumoricidal capacity and its stimulation by endotoxins

Without known stimulation in vivo and in vitro, resident peritoneal macrophages from 5 conventional or specific pathogen-free (SPF) rat strains [Hairless (H), BDIX, Wistar (W), Sprague-Dawley (SD) and Long-Evans (LE)] exhibited an in vitro strain-dependent cytolysis against DHD-K12/TS cancer cells. This natural cytolysis was also observed when polymyxin B was added to the culture medium. The percentage of natural cytolysis varied from one rat to another but was significantly different according to the strain. In the presence of 10 micrograms endotoxin/ml, macrophages from BDIX, W, SD and LE rats were always cytolytic, whilst those of H rats were irregularly cytolytic. Endotoxins induced or increased macrophage-mediated cytolysis from H, BDIX, W and SD rats, but they were without effect for LE rats. The endotoxin effect depended on the level of natural cytolysis. In contrast to mouse resident peritoneal macrophages, which were not naturally cytolytic and not activated in vitro by endotoxins, these results show that rat resident peritoneal macrophages can be naturally cytolytic. This cytolysis can be enhanced by endotoxins as the sole in vitro stimulus. Rat macrophage natural cytolytic activity is strain-dependent.     

Mitogen responses and interferon production after exposure of human macrophages to infectious and inactivated influenza viruses

Human macrophages were exposed to two influenza A viruses representing different subtypes. The donors were likely to have been exposed to one subtype (H3N2) but not to the other (H0N 1). Similar effects upon the macrophages were observed for both subtypes: macrophage enhancement of mitogen-stimulated lymphocyte transformation responses was depressed, and the macrophages produced interferon. In contrast, macrophages exposed to inactivated virus exhibited normal enhancement of lymphocyte transformation response, yet produced interferon, although in lower titers than did macrophages exposed to infectious virus.     

Effects of acetate on the immune system of mice

The effects of acetate on antibody production and cell-mediated immunity in mice were investigated. Polyclonal antibody responses could be enhanced in vivo by single intraperitoneal administration of acetate (5 mg/mouse) in C57BL/6 mice but not in DBA/2 mice. No enhancement of antibody production by acetate was also induced in athymic C57BL/6 nude mice and carrageenan-pretreated, macrophage-depleted mice. The inoculation of acetate-nonresponder BDF1 mice with T-cells and peritoneal adherent cells derived from acetate-treated C57BL/6 mice resulted in an enhanced antibody response. These results suggest that acetate increases polyclonal antibody responses in vivo by activating indirectly T-cells and macrophages. Acetate administration increased delayed hypersensitivity to pircryl chloride in C57BL/6 mice but not in DBA/2 mice. Allogeneic mixed lymphocyte reaction (MLR) of T-lymphocytes derived from the spleen of acetate-treated C57BL/6 mice was also enhanced. The natural killer (NK) activity and antibody-dependent cell-mediated cytotoxicity (ADCC) were also increased in C57BL/6 mice that were administered acetate. The possible mechanism of the immunopotentiating effect of this chemical is discussed.     

Synergistic effect of liposomes and endotoxins on the activation of rat macrophage tumoricidal activity

Macrophage biological responses to endotoxins have been extensively studied; nevertheless, the mechanisms by which endotoxins activate macrophage tumoricidal activity are not currently understood. We used liposomes to investigate the interaction of endotoxins with macrophages. In a medium containing 10 micrograms endotoxin/ml, macrophage-mediated cytolysis ranged from -7 to 36%. In all the experiments, 1mM dipalmitoyl phosphatidyl choline (DPPC) small unilamellar liposomes significantly induced or enhanced cytolysis, ranging from 30-90%. Liposomes and endotoxins had a synergistic effect on the macrophage cytolytic activity. This effect was dose-dependent on liposome concentration, ranging from 0.25-1 mM or 2 mM. Liposomes decreased the endotoxin concentration threshold necessary to induce cytolysis. They did not modify the kinetics of macrophage activation. Liposomes did not modify the binding of tumor cells to macrophages. The optimum synergistic effect was obtained when liposomes were present during the first 18 h of the mixed culture of macrophages and target cells, before adding endotoxins for the next 18 h. When cholesterol was added to DPPC (M/M), liposomes did not enhance but rather inhibited macrophage activation by endotoxins.     

Effects of Trypan Blue Treatment on the Immune Responses of Mice

It has been reported that trypan blue treatment decreases the nonspecific resistance of mice to transplanted tumors and inhibits the in vitro cytotoxic activity of activated macrophages. We wished to determine whether this effect of trypan blue could be due to a selective inhibition of certain macrophage functions or whether it reflected a broader form of immunosuppression. We therefore tested the effects of trypan blue on a variety of immunological responses. Treatment of mice with trypan blue delayed their rejection of skin allografts and transplants of a highly antigenic syngeneic ultraviolet light-induced tumor. Trypan blue treatment of either donor or recipient decreased the local graft-versus-host reaction. Filtration of lymph node cells from trypan blue-treated donors on a nylon wool column before use in the graft-versus-host assay abrogated the depressive effect of trypan blue. A transient reduction in the blastogenic response of spleen cells to concanavalin A and lipopolysaccharide mitogens was observed after a single injection of trypan blue, but the response of lymph node cells was unaffected. The depressed response of splenic lymphocytes was not entirely reversed by removal of adherent cells. The primary and secondary hemagglutinin responses to sheep erythrocytes were unaffected in trypan blue-treated mice, and the proportion and phagocytic activity of thioglycolate-induced peritoneal macrophages were also unaltered. We conclude that treatment of mice with trypan blue selectively inhibits certain macrophage functions but, at high doses, it can also inhibit some lymphocyte activities.     

Cytolytic activity of macrophages from 5 strains of rats

Resident peritoneal macrophages from 5 conventional or pathogen-free rat strains, without in vivo or in vitro stimulation, were generally tumoricidal in vitro against tumor cells: this spontaneous cytolysis rate varied from one rat to another but was significantly different according to the strains. Endotoxins induced or increased macrophage-mediated cytolysis from BDIX, Wistar and Sprague-Dawley rats; they were without effect on those from Long-Evans rats and their effect was irregular on Sprague-Dawley Hairless macrophages. Endotoxin effect depended on natural cytolysis intensity.     

Effect of Ly 5 allotype on in vitro immune responses.

We have tested in vitro immune responses of several kinds to determine if Ly 5 allotype influences reactivity of murine splenocytes in processes thought to involve the T200 glycoprotein. Matings were established among C57BL/6J (B6) (Ly 5.1) and C57BL/6-Ly 5.2 (B6-Ly 5.2) congenic mice (both H-2b) to obtain sibling mice segregating for alloalleles of the Ly 5 system. F2 progeny of the three Ly 5 genotypes were tested for antibody-dependent cell-mediated cytotoxicity (ADCC), proliferation in allogeneic mixed lymphocyte culture (MLC), mitogen responsiveness, natural killer (NK) cell activity, and cytotoxic T-lymphocyte (CTL)-mediated cytotoxic activity. We observed that in Ly 5 segregant mice, higher CTL activity was associated with the Ly 5.2 type. No allotype effect was observed in MLC, mitogen responses, and NK cell-mediated cytolysis. In the parental and F1 animals, mice carrying the Ly 5.2 allele had significantly higher ADCC levels, though this effect was not seen in the segregants. Our results indicate that Ly 5 or some closely linked gene or genes influence CTL activity of murine splenocytes in vitro.     

The nonspecific helper effect of mixed lymphocyte reactions on the induction of T cell-mediated immunity in vitro

We compared induction of sensitization in the mixed lymphocyte reaction (MLR) and in a mouse anti-fibroblast reaction. Our study demonstrated that induction processes in these two reactions, both known to be T cell-mediated reactions, are governed by different factors. Moreover, these two reactions interact with each other and this interaction is manifested by the ability of a MLR occurring during the sensitization phase of the anti-fibroblast reaction to enhance specific cytolysis measured during the effector phase. This helper effect appears to be immunologically nonspecific, since it can be obtained by the use of third party, antigenically unrelated lymphocytes as stimulator cells in the MLR.