Enumeration of immune interferon-producing cells induced by allogeneic stimulation.

We succeeded in enumerating interferon-producing cells induced by allogeneic stimulation, and proved that they were indeed T lymphocytes. The peak level of these cells in spleen was attained on day 5 after immunication, was maintained for about 2 days, and declined thereafter. Titers of interferon produced in vitro by sensitized spleen cells were maximum on day 7. This suggests that the maturation of immune interferon-producing cells follows cell proliferation after antigen stimulation.     

Generation and Maintenance of Immune Interferon-Producing Cells Induced by Allogeneic Stimulation in Mice

When spleen cells derived from C57BL/6 mice immunized with L cells 7 days previously were cocultured with antigenic cells, immune interferon appeared in the culture fluid. We analyzed the tissue distribution of the immune interferon-producing cells (IIPC) which appeared in various lymphoid organs after allogeneic stimulation. Although fluid from cocultures of L-cell-sensitized thymocytes and L-cells could not detect interferon activity consistently, small numbers of IIPC could be detected by using the enumeration method of IIPC. The generation, maintenance, and nature of IIPC emerging in the spleen were different depending on how the host mice were immunized. Multiple antigenic stimulations were more effective and induced longer-lasting immune interferon production than a single stimulation. IIPC induced by a single stimulation appeared to be sensitive to cortisone, vinblastine, and cyclophosphamide and were relatively short lived. In contrast, IIPC induced by multiple stimulations seemed to be partially resistant to these drugs and long lived. When mice were immunized with intact L-cells, carrageenan, a known antimacrophage agent, had no effect on immune interferon production. However, when mice were immunized with solubilized L-cell antigen, this drug displayed a suppressive effect on immune interferon production.     

Gamma interferon production in allogeneic stimulation: antigenicity that is sufficient to cause interferon production.

Spleen cells obtained from allogeneic cell-primed mice produced immune interferon when cocultivated with the antigenic cells. The purpose of this study was to clarify the antigenic determinants for immune interferon production in this allogeneic stimulation system. An incompatibility at the K end alone or at the D end alone of the H-2 complex was sufficient for immune interferon induction. No interferon production was observed in the combinations between strains of mice that differ for the non-H-2 regions, including the M locus. Immune interferon-producing cells (IIPC), induced by difference of the H-2K or H-2D regions, recognized the specificities controlled by the H-2K or H-2D regions, respectively; namely, there was no cross-reaction between the two regions. The difference between H-2d and H-2b did not cause interferon production in the combinations of which non-H-2 regions were very similar to each other. IIPC were not induced in the combination, but when IIPC were properly induced in B6 spleen cells, the IIPC could recognize the specificities controlled by the H-2d region (B10D2) and produced immune interferon.     

Suppression of interferon production in mouse spleen cells by cytochalasin D.

Cytochalasin D is thought to impair microfilament function. The present study has investigated its effects on four different systems in which interferon is formed, namely (1) mouse fibroblasts induced with virus (2) mouse spleen cells induced with virus, or (3) with endotoxin or (4) by allogeneic stimulation. Cytochalasin D did not suppress formation of interferon by fibroblasts (L cells) or spleen cells stimulated with either HVJ or NDV. However it did suppress production of interferon by spleen cells in response to endotoxin or an allogeneic stimulation; here its action was apparently not on the secretion of interferon, but on some earlier event. It also suppressed the production of interferon by mouse spleen cells induced with HVJ if this had been u.v. irradiated for more than 15 min: this suggests that cytochalasin D sensitive structures do play some role in interferon production by mouse spleen cells when stimulated with HVJ, as well as when they are stimulated with endotoxin or an allogeneic stimulus.     

Production of gamma interferon in mice immune to Rickettsia tsutsugamushi.

C3H/He mice immunized by subcutaneous infection with Rickettsia tsutsugamushi Gilliam were examined for the production of immune interferon after intravenous administration of irradiated strain Gilliam antigen, in supernatants of immune lymphocytes stimulated with specific antigen, and after a secondary challenge with viable rickettsiae. Mice administered various doses of irradiated whole-organism antigen 28 days after immunization showed circulating levels of interferon which peaked 4 h after inoculation and were antigen dose dependent. The interferon produced was pH 2 sensitive and stable at 56 degrees C for 1 h and was neutralized by antiserum directed against immune, but not against alpha/beta, interferon. The production of another lymphokine, macrophage migration inhibition factor, paralleled that of interferon. The interferon produced by cultures of spleen cells obtained from immune animals was antigen specific and dose dependent. Peak levels were obtained 48 to 72 h after the addition of antigen. The interferon produced by spleen cell cultures after stimulation with Gilliam antigen was characterized as immune interferon by the same physical and antigenic criteria used for serum interferon. Interferon was produced in vitro by the Thy-1.2+ lymphocyte and required the presence of a spleen-adherent cell population. Immune mice produced high circulating levels of immune interferon after intraperitoneal challenge with viable rickettsiae, which suggested a possible role for interferon in the resistance of immune mice to rechallenge with R. tsutsugamushi.     

Interferon production in L cells persistently infected with hemagglutinating virus of Japan (HVJ).

The present study shows that when L cells persistently infected with hemagglutinating virus of Japan (HVJ) (L-HVJ cells) were incubated at 32°, the interferon-producing capacity of the cells was suppressed and was restored by the temperature shift-up to 38°. Synthesis of envelope protein antigen was not detected in the L-HVJ cells incubated at 38° which could produce interferon. Moreover, glutamine-starved L-HVJ cells did produce interferon even at 32°. The relationship between the suppressed state of interferon production and synthesis of viral envelope protein is discussed.     

Reverse plaque formation by hog cholera virus of the GPE-strain inducing heterologous interference.

The general capacity of germfree mouse spleen cells to produce interferon in vitro in response to various stimuli was investigated. The interferon response of germfree mouse spleen cells in vitro, when compared with that of the conventionals, appears to be lower to some inducers. Interferon production in vitro stimulated by hemagglutinating virus of Japan (HVJ) or BHK-HVJ cells (BHK cells persistently infected with HVJ) was apparently suppressed in germfree mouse spleen cells as compared with the corresponding conventionals, whereas no difference of interferon production was observed between germfree and conventional mouse spleen cells in response to Newcastle disease virus, Escherichia coli endotoxin, poly(I:C), and phytohemagglutinin. Although monocontamination with HVJ had no enhancing effect on the interferon-producing ability of germfree mouse spleen cells in response to HVJ, conventionalization for 2 weeks greatly enhanced interferon-producing capacity.     

Effect of “immune” RNA on cells of different types interacting in the immune response

Simultaneous injection of bone marrow antigenic cells incubated with “immune” RNA, thymus cells, and sheep's red cells into irradiated CBA or DBA/2 mice leads to greater accumulation of antibody-forming cells in the recipients' spleen than in mice injected with intact bone marrow cells or the same cells incubated with normal RNA. Pre-incubation of thymus cells with “immune” RNA does not cause any change in the accumulation of antibody-producing cells. Incubation of peritoneal exudate cells with “immune” or normal RNA like-wise had no effect on the character of accumulation of antibody-forming cells.     

Lymphocyte bacterial rosette test: methodological details and effect of metabolic inhibitors and divalent cations

Lymphocytes bind certain bacteria. This property has been utilized in the lymphocyte bacterial rosette assay to identify T and B cell subsets. Here we determined the optimum conditions for the assay, studied the effect of metabolic inhibitors and divalent cations and compared bacterial rosette-forming lymphocyte subpopulations with those defined with monoclonal antibodies. The strains used were Brucella melitensis, Bacillus subtilis, Staphylococcus aureus and Escherichia coli. Optimum attachment was obtained at 4 degrees C in 6% BSA with ultrasonicated bacteria. Pretreatment of lymphocytes with the microfilament-disruptive drug cytochalasin B suppressed the binding of bacteria, whereas colchicine (inhibitor of microtubules), puromycin (inhibitor of translation), sodium azide (inhibitor of oxidative phosphorylation) and 2-deoxyglucose (inhibitor of glycolysis) had no effect. Divalent cations were required for the attachment of bacteria. B. melitensis bound to DR-positive cells, whereas the other bacterial strains rosetted OKT4- OKT8- and DR-positive cells without exhibiting helper or suppressor T cell or B lymphocyte specificity.     

Regulation by T cells of delayed hypersensitivity reaction in mouse lung as reflected by mononuclear cells, mast cells and mucus-producing cells

The appearance of mononuclear cells, mast cells and mucus-producing cells in the lung and their linkage to the development of delayed hypersensitivity (DH) reactions were studied. Adoptive transfer of immune lymph node cells, spleen cells and serum and in vivo treatment with monoclonal antibodies to L3T4-positive T cells in Balb/c mice were performed to investigate the cellular regulation of the number of mononuclear cells, mast cells and mucus-producing cells in the lung. Immune lymph node cells and, to a lesser extent, immune spleen cells from mice sensitized epicutaneously with picrylchloride transferred DH reactions to the recipients as assessed by ear thickness increase after challenge. Serum from sensitized mice was not able to transfer a DH reaction. Cyclophosphamide treatment of donor mice increased the DH reaction in the recipient mice. Adoptive transfer of immune lymph node cells and spleen cells gave a slight increase in the number of mononuclear cells in the lung of recipient mice compared with controls. This weak accumulation of mononuclear cells in the lungs of recipient mice, however, was not accompanied by a consistent increase in the number of mucus-producing cells and mast cells. The number of spleen cells expressing the L3T4 antigen decreased after in vivo treatment with the monoclonal GK1.5 (anti-L3T4) antibody as assessed by immunohistochemistry. This antibody treatment also resulted in an inhibition of the DH reaction and a decrease in the number of mononuclear cells and mucus-producing cells, but not in mast cells in the lung of sensitized and challenged mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Requirement for continuous antigenic stimulation in the development and differentiation of antibody-forming cells. Effect of antigen dose

The concept that antigen has a continuous role in the recruitment and differentiation of immune progenitor cells was tested with optimum and suboptimum doses of heterologous erythrocytes in mice. These studies further evaluated an immune cell maturation scheme in which continuous antigenic stimulation is required for both the recruitment of `antigen-sensitive units' and the expansive proliferation of a distinct sensitized cell compartment, which undergoes irreversible differentiation to functional antibody-forming cells. Haemolytic plaque-forming cell capacity during both the primary and secondary immune reactions were studied, both in the intact animal and with the spleen cell transfer technique. This in vivo culture technique was used to measure the sensitized cell compartment in the absence of existing antibody regulatory mechanisms. The results clearly demonstrate a higher detectable secondary immune capacity in the suboptimum antigen dose group than in the optimum antigen dose group. This was demonstrated for both the 19S and 7S cellular responses, as well as with humoral antibody levels measured in the spleen cell recipient mice. It can be concluded that in the presence of a suboptimum dose of antigen, which rapidly diminishes during the early intervals of the primary response, there is adequate recruitment with subsequent preservation or rescue from antigen-mediated depletion of the sensitized cell compartment, at the expense of the detectable primary response.     

Analysis of the lytic step in the herpes simplex virus antibody-dependent cellular cytotoxicity system

An antibody-dependent cellular cytotoxicity (ADCC) system in which herpes simplex virus-infected Chang liver cells are used was assessed for its dependency on cellular energy, ribonucleic acid and protein synthesis, and cytoskeletal structures such as microfilaments and microtubules. The cytotoxic reaction was only slightly inhibited when glycolysis was blocked in a glucose-free medium containing 10(-2) M 2-deoxy-d-glucose. It was more substantially inhibited when respiration was blocked with 10(-2) M sodium azide. The reaction was totally ablated, however, only when both glycolysis and respiration were suppressed. This inhibitory effect of energy deprivation was mediated solely at the level of the effector cell. Ribonucleic acid synthesis or protein synthesis by the effector cells was not required, as shown by the fact that neither actinomycin D, cycloheximide, nor emetine significantly inhibited ADCC. The ADCC reaction was partially inhibited by cytochalasin B, whose inhibitory effect was rapidly reversible, and was completely and irreversibly inhibited by cytochalasin A. Cytochalasin A acted on the effector cells rather than the target cells. The reaction was also partially inhibited by colchicine, whose inhibitory effect was directed solely against the effector cells and was slowly reversible. The inhibitory effects of cytochalasin B and colchicine, when used in tandem at submaximal inhibitory concentrations, were slightly more than additive. The results suggest a cooperative role for effector cell microfilaments and microtubules in mediating ADCC. Kinetic studies of ongoing herpes simplex virus ADCC reactions after initial centrifugation showed that the lytic step requires expenditure of metabolic energy as well as intact function of both microfilaments and microtubules. These findings, in concert with previous data, indicate that the ADCC process against herpes simplex virus-infected Chang liver cells can be resolved into adhesion and lytic steps. The lytic step can be readily distinguished from the adhesion step by its increased sensitivity to low ambient temperature or metabolic energy deprivation, its sensitivity to thermal inactivation, its requirements for extracellular divalent cations, and its dependence on normal function of both microfilaments and microtubules.     

In vitro culture of coxsackievirus group B, type 3 immune spleen cells on infected endothelial cells and biological activity of the cultured cells in vivo.

Spleen cells from male BALB/c mice infected 7 days earlier by an intraperitoneal injection of 3 X 10(4) PFU of a myocarditic strain of coxsackievirus B-3 lysed virus-infected endothelial cells in a 51Cr release assay. Cytotoxic activity in the in vivo sensitized spleen cell population could be further increased by culturing the immune spleen cells from infected mice on virus-infected or uninfected endothelial cells for 6 to 7 days in vitro. Cytotoxicity of in vitro cultured spleen cells to infected targets was mediated by T lymphocytes since reactivity was abolished by treatment of the spleen cells with anti-thy 1.2 serum and complement. Reciprocal assays with BALB/c and C57BL cells indicated that maximum cytotoxicity occurred when spleen cells were sensitized on syngeneic endothelial cells. Other experiments showed that spleen cells sensitized to coxsackievirus B-3 or encephalomyocarditis virus were selectively cytolytic to targets infected with the homologous virus. Adoptive transfer of T cells cultured in vitro on infected endothelial cells retained their ability to induce myocarditis in T-lymphocyte-deficient mice.     

Role of macrophages in tumour immunity: II. Involvement of a macrophage cytophilic factor during syngeneic tumour growth inhibition*

Growth inhibition by immune or armed macrophages of target L5178Y or SL2 (DBA/2) lymphoma cells leading to their death required an immunologically specific cell-to-cell contact, and was not mediated via a soluble product of the macrophages. The lymphoma cells were not irreversibly damaged if left in contact for up to 24 hours with the immune macrophages and grew normally when removed, but while in contact there was little or no growth of the cells. Immune macrophages progressively lost their cytotoxic capacity after prolonged exposure to the target cells. The antigenic recognition of specific lymphoma cells by immune macrophages could be blocked by the presence of allo-immune serum directed against the lymphoma cells, but this was not affected by treatment of the macrophage monolayers with rabbit anti-mouse γ-globulin, and only slightly affected by high concentrations of trypsin. The involvement of cytophilic factors during the cytotoxic reaction is discussed in relation to the presence of γ-globulin or non-immunoglobulin factors on immune macrophage membranes and after the arming of non-immune macrophages by contact with hyperimmune spleen cells or with SMAF (the specific macrophage-arming factor) found in supernatants of mixed cell cultures of sensitized lymphoid cells and specific lymphoma cells.     

Immune interferon induced by phytohemagglutinin in nude mouse spleen cells.

Phytohemagglutinin is able to trigger interferon synthesis in spleen cell cultures from nude (nu/nu) mice as effectively as in splenic cell cultures from haired, control (nu/+), thymus-bearing mice. A minor theta-bearing cell population present in the spleen of nude mice appears essential to phytohemagglutinin interferon production, although cooperating cells are also required. The properties of nude mouse phytohemagglutinin interferon are indistinguishable from those displayed by the interferon induced in thymus-bearing mouse spleen cell cultures. Both interferons are unstable at pH 2 and cannot be neutralized by an antiviral interferon serum; hence, their characteristics correspond to those described for type T interferon. As in the case of viral interferon, pretreatment of L cells with nude phytohemagglutinin interferon induced specific enhanced phosphorylation of a 67,000-molecular-weight protein in vitro when cell extracts were incubated with double-stranded RNA and gamma-[32P]ATP.     

In vitro production and cellular origin of murine type II interferon.

Antigen-specific type II interferon was produced in vitro by harvesting supernatants of spleen cell cultures from Swiss-Webster mice sensitized with Mycobacterium bovis strain BCG and challenged with old tuberculin. Treatment of C3H mouse spleen cell cultures with appropriate anti-Ia, anti-IgG, anti-Thy-1 or anti-Ly-2,3 sera resulted in a significant decrease in production of type II interferon. Removal of nylon wool adherent cells or cells with histamine receptors by column chromatography similarly caused reduced production of type II interferon. Recombination of spleen cell cultures treated with anti-Ia and anti-Thy-1 sera or of cells treated with anti-IgG and anti-Thy-1 resulted in restored production of type II interferon. Interferon production was also restored by combination of cells passed through histamine columns with anti-Ia treated cells, or those passed through nylon wool columns with anti-Thy-1 treated cells. Anti-Ly-1 serum treatment had no effect on interferon production. Removal of plastic-adherent cells or cells that had phagocytosed carbonyl iron also decreased interferon production, suggesting that macrophages were also involved in type II interferon production. Recombination of non-adherent spleen cells with anti-Ia and anti-Thy-1 sera treated spleen cells, however, did not restore interferon production, suggesting that other cells in addition to macrophages are depleted by the adherence procedure. These findings indicate that type II interferon is produced by suppressor or cytotoxic (Ly-2,3+) T lymphocytes in co-operation with one or two additional cell types: (i) B lymphocytes, and (ii) macrophages.     

Human neutrophil aggregation and increased adherence to human endothelial cells induced by heat-aggregated IgG and immune complexes.

Several types of IgG-dependent phagocytic stimuli independent of complement were investigated for their property to induce human polymorphonuclear neutrophil leucocyte (PMN) aggregation and adherence to human endothelial cells (EC) in culture. A Coulter counter method was employed for the detection of cell aggregation. Aggregated IgG, ovalbumin-anti-ovalbumin (OV anti-OV) immune complexes (both insoluble and soluble) and opsonized latex particles induced a significant degree of PMN aggregation which was detectable as early as 2 min after exposure of PMN to these stimuli. This aggregation was dependent on divalent cations (Ca++, Mg++). The same phagocytic stimuli furthermore significantly increased adherence of PMN to cultured human EC and serum-coated plastic. Controls consisting of native IgG, and OV anti-OV complexes prepared from Fab')2 antibody failed to induce either aggregation or increased adherence of PMN. These data suggest that exposure of PMN to IgG-dependent phagocytic stimuli induces increased adhesiveness of PMN and that interaction between the Fc-receptor of PMN and the Fc-portion of phagocytic stimuli is essential for this effect.     

Penicillamine and penicillin can generate antigenic determinants on rat peritoneal cells in vitro.

Conjugation of the protein-reactive drugs D-penicillamine (PA) and benzylpenicillin (BP) to immune cells to generate drug-derived antigenic determinants has been implicated in drug-induced allergies and autoimmunity. We have therefore developed an in vitro system to demonstrate and characterize the formation of cellular antigens by these drugs. Binding of PA and BP to rat peritoneal exudate cells was detected by a cell ELISA, employing rabbit antisera specific for each drug, and an indicator system employing a second antibody coupled to biotin-streptavidin-beta-galactosidase. For both drugs, binding was detected over the concentration range 125-1000 micrograms/ml. PA bound cells rapidly (maximum binding within 10 min), whereas BP bound relatively slowly (maximum binding occurring later than 4 hr). A possible role for intracellular processing and cellular metabolic activity in the generation of these drug-derived antigenic determinants was examined. Pretreatment of the cells with the fixative paraformaldehyde significantly enhanced binding of PA but not BP. Treatment of cells with the lysosomotropic agents ammonium chloride or chloroquine, or with the metabolic inactivator sodium azide, did not affect the binding of either drug compared with untreated control cells. However, treatment with the oxidising agent copper sulphate, or the cellular activator phorbol myristate acetate, did significantly enhance binding of both drugs to the cells. Therefore, binding of PA and BP to the cell surface appears not to require an intracellular processing event to generate a recognizable antigenic determinant, but is enhanced by treatments that stimulate oxidative processes.     

Cell-mediated cytotoxicity against ectromelia virus-infected target cells. II. Identification of effector cells and analysis of mechanisms

Cell-mediated cytotoxicity of ectromelia-immune spleen cells against ectromelia-infected L929 target cells was abrogated by treatment of the spleen cells with anti-Θ antibody and complement. Treatment of spleen cells with anti-immunoglobulin serum and complement, or removal of macrophages had little or no effect on cytotoxicity. Specific or nonspecific soluble cytotoxic factors were not detected, and exogenous interferon did not enhance cytotoxicity. No blocking of cytotoxicity was detected with either hyperimmune or 9-day immune anti-ectromelia serum. By varying the spleen cell-target cell ratio from 3: 1 to 400: 1, it was shown that the efficiency of cytotoxicity was inversely related to spleen cell density. These results were interpreted to mean that thymus-derived (T) cells, probably acting alone, were responsible for cytotoxicity and that the mechanism involved contact between T cell and target cell.     

Natural killer cells and interferon

Natural killer cells are cytotoxic lymphocytes found in all examined vertebrates and implicated as mediators of tumor surveillance in experimental animal models. Normally cytotoxic for a selective group of tumor target cells, NK cells can be dramatically activated to a level of high cytotoxicity by interferon and interferon-inducing agents, such as viruses, bacteria, lectins, and synthetic polynucleotides. These interferon-activated cells differ physically and antigenically from nonactivated precursor cells. All types of interferon (alpha, beta, and gamma) are capable of activating NK cells. Conversely, interferon treatment of target cells renders them resistant to NK cell-mediated cytotoxicity. Tumor cells induce interferon synthesis in leukocyte cultures, and the interferon-producing cell has properties in common with NK cells. The complexities of these effector cell-target cell-interferon interactions will be discussed.