The multi-factorial pathogenesis of autoimmune disease.

Development of organ-specific autoimmune diseases depends on both an abnormal immune regulation and a genetically determined primary susceptibility of the target organ to the autoimmune attack. In addition to the essential genetically determined prerequisites there are also facultative, modulating factors that influence the outcome of an autoimmune disease. This concept is exemplified in the Obese strain (OS) chicken model which develops a spontaneous autoimmune thyroiditis closely resembling human Hashimoto disease. Three modulating factors are specifically addressed, viz. (a) the lower threshold of OS thyroid epithelial cells for the gamma-interferon-induced MHC class II antigen expression as compared to normal controls, (b) the decreased glucocorticoid tonus of the OS and (c) the presence of a new endogenous virus (ev 22) locus in the OS that has so far not been found in any normal strain and which seems to influence the glucocorticoid-mediated immunoregulatory process.

Comparison of roles of serine esterase in chicken and human natural cytotoxicity.

The requirements for serine esterase activity in the spontaneous cell-mediated cytotoxicity of human lymphocytes and chicken granulocytes have been compared. The lysis of K-562 target cells and of LSCC-H32 chicken target cells was prevented by the serine esterase inhibitor TPCK. ATEE, the substrate of chymotrypsin, impaired both cytotoxic reactions, but to a lesser degree the cytotoxicity of chicken granulocytes. TPCK inhibited the "trigger" mechanism in an early calcium-dependent step and later calcium-independent events in both systems. However, calcium-independent lysis was depressed by serine esterase inhibitor only in the avian cytotoxicity. These findings suggest that avian target cell cytolysis consists of similar sequential phases to those already demonstrated in the human NK cell reaction, and serine esterase is required during several stages of cytotoxicity in the avian system.

Are granulocytes the main effector cells of natural cytotoxicity in chickens?

Purified peripheral blood granulocytes from chicken were tested for cytotoxic activity against two types of virus-transformed chicken cell line, LSCC-H32 and LSCC-RP9. Strong cytotoxicity could be demonstrated, as measured in a 4-hr 51Cr-release assay, especially against the fibroblastoid LSCC-H32 cells. The degree of cytotoxicity was dependent on the E:T ratio. Normal CEF cells were completely resistant to the cytotoxicity. No cytotoxicity of human granulocytes could be observed against a variety of adherent and non-adherent target cells, as measured by the same microcytotoxicity technique. The priority of granulocytes in the natural cytotoxicity in the avian system is, therefore, suggested.

The role of interferon in the adenovirus-induced augmentation of granulocyte-mediated cytotoxicity in chicken.

The effects of human adenoviruses on the granulocyte-mediated natural cytotoxicity of chicken leukocytes were investigated. A significant, but transient augmentation of granulocyte cytotoxicity was observed 24 h after virus injection, followed by a relatively long period of its suppression. A good correlation was found between the augmented cytotoxicity and interferon induction. The interferon-inducing capacity of adenovirus type 6 and type 12 in vitro similarly ran parallel with their ability to stimulate granulocyte-mediated cytotoxicity. An adenovirus-induced elevation of cytotoxicity was not observed when IFN production was inhibited by pretreatment of the leukocytes with monoclonal antibody specific for bursal cells and monocytes. In addition, anti-IFN antibody abrogated the stimulation of cytotoxicity as well. During the in vitro experiments in which granulocyte-specific monoclonal antibody was applied, evidence was found that the effector cell activity is associated with the granulocytes. These results suggest that both the in vitro and the in vivo adenovirus-induced augmentation of granulocyte-mediated cytotoxicity is due to the IFN-inducing capacity of the virus. In chickens, the rapid augmentation of the granulocyte cytotoxicity may be important in the acute stage of infection, increasing the resistance to the virus in question and also to bacterial infections.

Granulocyte-specific monoclonal antibody inhibiting cytotoxicity reactions in the chicken.

Monoclonal antibody (MAb) 1C3, specific for chicken granulocytes, is described for the first time. Treatment of peripheral blood leukocytes with this MAb markedly decreased natural cytotoxicity reaction against the target cell line. The antibody-dependent cellular cytotoxicity (ADCC) activity of purified granulocytes was also severely affected by treatment with MAb 1C3. These results suggest that 1C3 detects a functional surface antigen on chicken granulocytes and support the hypothesis that granulocytes are the main effector cells in natural cytotoxicity in the chicken.

Synergistic action of quercetin and murine alpha/beta interferon in the treatment of Mengo virus infection in mice.

ABD2F1 mice were infected intraperitoneally (i.p.) or intranasally (i.n.) with Mengo or Sindbis virus and treated with either crude murine alpha/beta interferon (MuIFN-alpha/beta) or quercetin, or both. MuIFN-alpha/beta given i.p. or intramuscularly (i.m.) 1-3 h before the infection had a dose-dependent protective effect regardless of the route of administration. When given after the infection, IFN did not show any effect. Oral quercetin, capable of protecting cardio, i.e. Mengo virus-infected mice, failed to show antiviral efficacy in Sindbis virus-infected animals. Of various combinations of quercetin and MuIFN-alpha/beta, a certain well defined regimen resulted in a significant enhancement of protection in Mengo, but not Sindbis, virus-infected mice. A marginally effective treatment regimen of quercetin (20 mg/kg, given 12 h before Mengo virus infection, and 10 mg/kg given both 1 h before and 12 h after infection) potentiated the activity of a single dose of MuIFN-alpha/beta (5000 IU 3 h prior to infection), giving 85-100% survivors compared to 50% for MuIFN-alpha/beta when applied alone (p less than 0.001).

The effect of interferon on chicken spleen lymphocytes.

The inhibitory activity of chicken interferon has been investigated on phytohemagglutinin (PHA)-induced DNA synthesis of chicken spleen lymphocytes inoculated with type 12 adenovirus and was purified by selective adsorption on Na-Al-silicate. As a control, the culture fluid of chicken leukocytes purified in the same manner was used. In some instances the control preparation also showed a slight inhibitory effect. To prove that the immunosuppressive activity of our interferon preparation was indeed due to its interferon content both the interferon and the control preparation were purified further by chromatography on Sephadex G 100 superfine gel. Only the fractions with antiviral activity of interferon preparation inhibited the PHA-response of the lymphocytes. The fractions with the same molecular weight of control preparation were without effect.

Inhibition of cytotoxicity of chicken granulocytes by serotonin and ketanserin.

Serotonin (5-HT) has been observed to impair the cytotoxicity of human natural killer (NK) cells. A study has now been made of the effect of 5-HT on the cytotoxic activity of chicken granulocytes. 5-HT at concentrations of 10(-3) to 10(-6) M inhibited the cytotoxicity of chicken granulocytes when added at the onset of the short-term cytotoxicity assay. Ketanserin, a 5-HT2 receptor antagonist, did not reverse the inhibitory effect of 5-HT on chicken granulocyte cytotoxicity. Moreover, ketanserin at concentrations of 5 x 10(-4) to 5 x 10(-7) M inhibited the cytotoxicity mediated by chicken granulocytes. Pretreatment of the effector cells for 2 h with chick fibroblast interferon reduced the inhibition of chicken NK cytotoxicity induced by 5-HT and by ketanserin. These data indicate that, in birds, the neurotransmitter 5-HT serves as a link between the central nervous system and the immune system, and that interferon can modulate the inhibitory effect of 5-HT on the function of cytotoxic granulocytes.

Antitumor activity of flavonoids on NK/Ly ascites tumor cells.

Among the various flavonoids, rutin and quercetin increased the survival time of mice inoculated with NK/Ly ascites tumor cells. The best results were obtained when the mice were given 2.0 mg rutin twice daily for 8 days. The O-silyl-substituted rutin and quercetin were less effective than rutin or quercetin themselves. Besides rutin, quercetin and morin two other flavonoids, luteolin and pelargonidin also exerted growth inhibitory effects on NK/Ly ascites tumor cells cultures in vitro.

Effect of human adenovirus on the ellipsoid-associated cells of the chicken's spleen.

Ellipsoid-associated cell (EAC) responded in two ways to the intravenously injected human adenovirus. The EAC may produce a chemotactic factor(s) which recruited heterophils to the periellipsoid white pulp. This early event was shown to occur immediately after the virus injection. Within 6 h of injection, the EAC disappeared from the ellipsoid and their vacated sites appeared edemic which suggested that the EAC may be the target cell of the adenovirus in the chicken spleen.

Impaired interleukin 2 production by spleen cells from mice infected with human adenovirus.

Spleen cells from mice infected with human adenovirus type 6 (Ad6) showed defective interleukin 2 (IL2) production 3-5 days after the infection. The response of spleen cells to exogenous IL 2 was also deficient. The impaired capacity of concanavalin A--(Con A)-activated spleen cells from Ad6--infected mice to utilize IL 2 seemed to be related to the depressed capacity of the infected splenocytes to express IL 2 receptors. The immunologic dysfunction following infection with Ad6 may be a consequence of deficiencies in both the elaboration of and response to IL 2.

Preparative separation of intact incomplete and empty adenovirus type 2 particles.

Complete adenovirus type 2 virions containing the whole genome were resistant to while empty capsids lacking DNA were breakable at a separation procedure involving customary CsCl gradient centrifugation. A purification method was developed using CsCl density gradient centrifugation in 5% glycerol which retained the integrity of incomplete particles as well as empty capsids. This procedure enabled to study the biological and physicochemical properties of different adenovirus type 2 particles.

Production and characterization of interferon induced in chicken leukocytes by concanavalin A.

Interferon (IFN) was induced in chicken peripheral blood leukocytes by exposure to Concanavalin A (Con A). It has reached a maximum level 96 hr after stimulation with the optimum dose of 10 micrograms/ml. The IFN was partially purified by chromatography on controlled-pore glass adsorbent and was characterized as a fairly acid-stable and heat-resistant, trypsin-sensitive and species-specific substance with Mr of 20,500 Da. Antiviral response by this type of IFN in chick fibroblast culture developed within several hours. This study provides first evidence of the presence of IFN in supernatants of mitogen-stimulated chicken peripheral leukocytes.

Involvement of prostaglandins in immunosuppression caused by adenovirus in mice.

The effect of intraperitoneally (i.p.) inoculated human adenovirus type 6 (Ad6) was tested for humoral immune response against sheep red blood cells (SRBC) in normal and indomethacin-treated mice, with the aim to elucidate the mode of the virus action in immunosuppression. The results indicate that inhibition of prostaglandin synthesis slightly influences the immunosuppressive effect of the virus. It is very likely that also other mechanisms are involved in the immunosuppression observed.

The role of macrophages in adenovirus-induced immunosuppression in mice.

A decreased humoral immune response to sheep red blood cells (SRBC) was demonstrated in CFLP mice inoculated with human adenovirus type 6 (Ad6 virus). The suppressive effect depended on the inoculation sites of the virus and SRBC. Inoculation by the same route, i.e. intraperitoneally (i.p.), resulted in a significant decrease of the humoral immune response to SRBC. When administering the virus and SRBC at different sites, no inhibitory effect could be detected. Immunosuppression was prevented by pretreatment of mice with silica depressing the macrophage function. The results indicate that peritoneal macrophages play an important role in the immunosuppression observed.

T antigen synthesis and resistance to interferon in human adenovirus type 12 infected chick cells.

Tye specific T antigen formation has been demonstrated in primary and secondary chick embryo cells (CEC) infected with adenovirus type 12. The frequency of cells synthesizing T antigen was closely dependent on the multiplicity of infection (MOI). At a MOI of 2.85 TCD50 per cell, T antigen was formed in 50% of cells. Cycloheximide inhibited T antigen formation while cytosine arabinoside had no such effect. CEC infected with adenovirus 12 produced interferon and T antigen, both appearing early and at about the same time of infection Exogenous chick interferon had no inhibitory effect on the formation of T antigen. In adenovirus 12-infected CEC, virus specific structural antigen could not be detected.

Failure of high doses of potentially antiviral agents to prevent death in virus-infected mice.

The influence of increasing doses of various potentially antiviral agents on Mengo virus-and Sindbis virus-infected mice was assessed determining death rates and histologic lesions. All drugs given in abundance showed dose-dependent decrease of antiviral activity following the maximum protective effects in either animal model. From the toxicological point of view the antiviral agents in question could be classified into two groups. High doses of tilorone hydrochloride or 10-carboxymethyl-9-acridanone produced toxic effects in both uninfected and virus-infected animals. In contrast, high doses of quercetin, double-stranded ribonucleic acid (dsRNA), 1-ethylisatin-S-n-butylisothiosemicarbazone or Norakin induced no obvious drug-mediated histologic alterations either in uninfected or in virus-infected individuals, but exerted a decreased protection in virus-infected animals. It is suggested that high doses of the compounds of second group adversely affect early host defence in both Mengo virus and Sindbis virus-infected mice.

Parvovirus contamination of a human type 12 adenovirus strain.

A strain of human type 12 adenovirus was found contaminated with virions 27 nm in diameter. After separation by membrane filtration, adsorption on to and elution from erythrocytes and heat treatment, the contaminant was classified as a parvovirus based on its biological and physico-chemical properties and virion morphology. This parvovirus failed to produce interferon and did not affect the interferon-inducing ability of the adenovirus from which it had been separated.

Effects of phorbol myristate acetate on interleukin-2 and accompanying interferon production of human leukocytes induced by heat-inactivated Staphylococcus aureus.

Interleukin-2 (IL-2) production induced by heat--inactivated Staphylococcus aureus (SAU) was enhanced by simultaneous addition of phorbol myristate acetate (PMA). The effect was optimal at a concentration of 10 ng/ml SAU; in the presence of 10 ng/ml PMA, the amount of SAU required for maximal IL-2 production was lower. The kinetics of SAU and of SAU plus PMA-induced IL-2 production were similar. Stimulated mononuclear cells produced interferon (IFN) in addition to IL-2. The titre of accompanying IFN was decreased in cultures stimulated with the SAU plus PMA combination. Plastic nonadherent sheep erythrocyte-positive cells were the most active in the SAU-induced IL-2 production. In contrast, the bulk of the IFN activity was produced by the nonadherent E rosette-nonforming cells. Neutralization of IFN with specific antibodies and pH 2 treatment indicated that SAU-induced IFN consisted mainly of alpha-IFN.

Differential effect of interferon on the production on virus- and mitogen-induced leukocyte inhibitory factors in human leukocyte cultures.

Interferon treatment of human leukocytes before stimulation with Sendai virus enhanced interferon synthesis but did not affect their leukocyte migration inhibitory factor (LIF) production. The same interferon pretreatment markedly enhanced both LIF and interferon productions when leukocytes were stimulated with concanavalin A.