Interferon action against reovirus: activation of interferon-induced protein kinase in mouse L929 cells upon reovirus infection

Experiments are presented which indicate that reovirus infection results in an activation of the interferon (IFN)-induced protein kinase in mouse L929 cells. This is indicated by (i) the phosphorylation in vivo of a 67,000 M_r (67K) polypeptide, which is characteristic of the IFN-induced protein kinase, within a few hours after reovirus infection of IFN-treated mouse L929 cells, and (ii) the phosphorylation in vitro of the 67K polypeptide as well as the α-subunit of exogenously added initiation factor eIF-2 in extracts of IFN-treated reovirus-infected cells without the addition of double-stranded RNA which is required in similar extracts from uninfected cells. In NIH 3T3 cells, which are deficient in (2′,5′)oligoadenylate-activated endonuclease, the replication of reovirus is inhibited by IFN treatment, though EMC virus replication is insensitive. It is suggested that this kinase activation observed upon reovirus infection may play a role in the antiviral action of IFN against reovirus.     

Mechanism of interferon action: Interferon inhibits the synthesis of viral proteins and induces protein P1 phosphorylation in both adenylate cyclase-deficient and cAMP-dependent protein kinase-deficient variants of Mouse lymphoma cells

The effect of interferon (IFN) treatment on the intracellular level of cyclic AMP, the synthesis of viral proteins and the induction of protein phosphorylation was examined in wild-type, adenylate cyclase-deficient (cyc^?), and cAMP-dependent protein kinase-deficient (kin^?) variants of mouse lymphoma S49 cells. Treatment with IFN or cholera toxin elevated the intracellular level of CAMP in wild-type but not in cyc^? cells. However, the synthesis of vesicular stomatitis virus polypeptides was inhibited to comparable extents by IFN treatment in all three types of S49 cells, wild-type, cyc^?, and kin^? Likewise, the phosphorylation of ribosome-associated protein P₁ was enhanced to a similar level in extracts prepared from IFN-treated wild-type, cyc^?, and kin cells. The results of this genetic analysis suggest that IFN may indeed elevate cAMP levels, but that the induction of an antiviral state and the P ₁ protein kinase by IFN in cell culture is not mediated by cAMP.     

Rabbit tumor necrosis factor: mechanism of action.

Rabbit tumor necrosis factor (TNF) was examined for effects on normal and transformed cells in culture. Several assays for killing of L-929 cell targets were developed, and their sensitivities were compared. Normal cells were not killed by TNF, and the discrimination between normal and transformed cells was shown not to be due to a cell cycle-dependent mechanism. TNF killing of L-929 cells was delayed for 10 to 12 h and thereafter showed concentration and time-dependent increases in cytolysis. Actinomycin D or cycloheximide treatment of L-929 cells resulted in an enhancement of the rate of cell killing as well as a shortening of the preceding lag period. TNF killing of L-929 cells was temperature dependent; cells were considerably more resistant to lysis at 25 degrees C and showed enhanced killing at 39 degrees C as compared to 37 degrees C controls. The slope of the dose curve showed less than single-hit kinetics. A model for cell killing whose general features incorporate both the specificity and catalytic properties of an enzymatic reaction is proposed for TNF action.     

The effect of interferon treatment of targets on susceptibility to cytotoxic T-lymphocyte killing: augmentation of allogeneic killing and virus-specific killing relative to viral antigen expression.

The effect on CTL lysis of treatment of CTL targets with IFNs has been investigated. Treatment of targets for alloreactive CTL with either IFN-alpha beta or IFN-gamma markedly augmented cytotoxicity. Cold competition experiments implied that CTL recognized the same target structure on both untreated and IFN-treated cells. This augmented lysis is presumably caused by IFN increasing expression of target MHC antigens. In the case of SFV-specific lysis of SFV-infected fibroblasts, IFN-alpha beta or IFN-gamma treatment somewhat reduced CTL lysis, but less so than Ab + C lysis which was abolished at moderate IFN concentrations; in the case of SFV-infected lymphoblastoid cells, CTL lysis remained the same or was slightly increased, whilst Ab + C lysis was reduced at moderate IFN concentrations and abolished at high IFN concentration; in the case of MSV/MLV-infected fibroblasts, CTL lysis was moderately increased whilst Ab + C lysis was decreased. IFN therefore increases virus-specific CTL cytotoxicity relative to viral antigen expression.     

Defective transport of hemagglutinin-neuraminidase glycoprotein of bovine parainfluenza-3 virus in interferon treated cell

Summary A defective transport of bovine parainfluenza-3 virus (PI-3V) hemagglutinin-neuraminidase (HN) glycoprotein was evidenced in interferon (IFN)-treated bovine turbinate (BTu) cells. Indirect immunofluorescence performed with monoclonal antibody to PI-3 HN glycoprotein demonstrated accumulation of this protein in the perinuclear cytoplasm of IFN-treated cells. Untreated, infected control cells had a generalized widespread fluorescence. Unfixed control cells showed a uniform surface fluorescence in contrast to a few specs of fluorescence on the plasma membrane of IFN-treated cells. Electron microscopic localization of HN protein was done by immuno-gold ultrastructural cytochemistry. Untreated cells had uniform gold label on the plasma membrane and around the budding virus particles with no label in the cytoplasm. In IFN-treated cells, however, there was an accumulation of gold particles in the cytoplasm with only a few particles on the cell surface. Quantitative analysis of HN protein on the cell surface by solid phase radioimmune-assay revealed a greater amount of this protein on the surface of control cells, than those on the IFN-treated cells.     

Negative regulation of rat natural killer cell activity by major histocompatibility complex class I recognition

The cytolytic activity of human and mouse natural killer (NK) cells is negatively regulated by self major histocompatibility complex (MHC) class I molecules on potential target cells. In the rat, protection by RT1 class I gene products has so far not been formally shown although the complex effects of foreign and self RT1 genes on polyclonal NK cell activity suggest that MHC recognition can have both stimulatory and inhibitory effects. Here we report that the expression of self-MHC class I molecules on target cells strongly inhibits lysis by a long term NK cell line derived from LEW (RT1^l) rats and by LEW NK cells activated by short-term culture in the presence of interleukin-2. This was demonstrated with mouse-rat hybridoma target cells expressing different rat MHC alleles and with mouse tumor target cells transfected with classical (RT1.A^l) and nonclassical (RT1.C^l) rat MHC class I genes. With hybridoma target cells, the strongest reduction in lysis as compared to the parental mouse myeloma line was observed when “self” (LEW) MHC was expressed, while hybridomas expressing other MHC alleles showed less and variable reduction. Transfection of RT1.A^l protected both L-929 fibroblasts and P815 mastocytoma cells from lysis by the NK cell line, while RT1.C^l only protected P815 cells, indicating that additional target cell properties regulate rat NK cell activity.     

Interferon enhances the amount of membrane-bound β2-microglobulin and its release from human Burkitt cells

Human leukocyte interferon (HuIFN-α) increased the amount of β2-microglobulin on the surface of human Burkitt lymphoma cells (Ramos) and also increased the amount released into the culture medium. The effect was observed 1 h after addition of IFN. These results suggest that the increase in β₂-microglobulin on the cell surface of IFN-treated cells is not due to a decreased shedding of antigen from the cell surface, nor an “unmasking” of surface antigen, but rather to an increased synthesis of antigen.     

Replication of sialodacryoadenitis virus in mouse L-2 cells

Summary Sialodacryoadenitis (SDA) is a naturally-occurring infection of the laboratory rat raused by the coronavirus, sialodacryoadenitis virus (SDAV). The study of SDAV has been limited because there is no widely available continuous cell line for the propagation of high titers of the virus. The purpose of this study, therefore, was to compare the ability of SDAV to replicate in the permanent cell lines, LBC, of rat origin, and the mouse cell lines. L-929 and L-2. Following 2 to 6 repeated passages of SDAV in LBC cells, the virus could be readily propagated in LBC and L-2 cells, but not in L-929 cells. Similarly, SDAV adapted to replicate directly in L-2 cells could be readily propagated in LBC, but not L-929 cells. In LBC and L-2 cells, cytopathic effect (CPE), viral antigen, viral particles, and virus infectivity could be demonstrated. Titers of up to 10^8.0 infectious viral particles/0.25 ml of culture fluid were obtained at 48 hours in L-2 cells. Titers in LBC cells were one to two logs lower. When susceptible rats were inoculated with eighth passage L-2 cell-adapted virus, they developed typical lesions of SDA. Virus could be recovered from infected tissues and propagated in L-2 cells on first passage. The ability to propagate SDAV to high titers in the widely available L-2 cell line should promote the study of this virus and facilitate its comparison with other murine coronaviruses.     

Siglec‐7 Defines a Highly Functional Natural Killer Cell Subset and Inhibits Cell‐Mediated Activities

Sialic acid‐binding immunoglobulin‐like lectin‐7 (Siglec‐7) is an inhibitory receptor expressed on natural killer (NK) cells. In this study, we investigated the relationship between Siglec‐7 expression and NK cell functions. Siglec‐7 was highly expressed on NK cells and was preferentially expressed by mature NK cells from peripheral blood of healthy adults. Siglec‐7⁺ NK cells displayed higher levels of activating receptors CD38, CD16, DNAM1, NKp30 and NKp46, but lower levels of inhibitory receptors such as NKG2A and CD158b, compared with Siglec‐7^– NK cells. Functional tests showed that Siglec‐7⁺ NK cells displayed more CD107a degranulation and IFN‐γ production than Siglec‐7^– NK cells. Siglec‐7 inhibited NK cell functions when interacting with specific antibodies. These data suggest that Siglec‐7 defines a highly functional NK cell subset and suppresses NK cell‐mediated functions when cross‐linked with specific antibodies.     

Inhibition of protein synthesis by vaccinia virus. I. Characterization of an inhibited cell-free protein-synthesizing system from infected cells

L-929 or HeLa cells infected with vaccinia virus in the presence of cycloheximide fail to resume protein synthesis upon removal of the drug 3.5 hr after infection. However, infected-treated LLC-MK2 cells resume protein synthesis upon removal of the drug. Cell-free protein-synthesizing systems prepared from such vaccinia virus infected-cycloheximide treated L-929 or HeLa cells, 30 min after removal of the drug, fail to incorporate amino acids in vitro in response to endogenous mRNAs, while similar extracts of LLC-MK2 cells are functional in vitro. The inhibition of protein synthesis is also characterized by a failure to respond to exogenous mRNAs (Globin, L cell poly (A), or EMC RNA). Such inhibited extracts have been separated into supernatant, ribosome, and ribosomal salt wash fractions. The response of ribosomes from infected and uninfected cells to EMC RNA in the presence of homologous and heterologous supernatant fractions shows that defects can be found in both ribosomes and supernatant fractions of infected-treated cells. Readdition of salt wash fraction of infected and uninfected ribosomes to the ribosome and supernatant fractions of infected and uninfected cells shows an additional lesion in the salt wash fraction of infected-treated cells. Addition of supernatant, ribosome, and salt wash fractions of infected and uninfected cells individually to reconstituted protein-synthesizing systems prepared from normal cells shows that the primary lesion in the infected-treated cells is the salt wash fraction of the infected ribosomes. This constitutes a crude initiation factor preparation and is consistent with previous observations that vaccinia virus-induced inhibition of protein synthesis is the result of a failure at the level of initiation.     

Target cell-dependent T cell-mediated lysis of vaccinia virus-infected cells

Vaccinia virus specific cytotoxicity against infected target cells was observed in vitro. Spleen lymphocytes from normal and immunized mice of the inbred strains C3H and DBA/2 were incubated with vaccinia virus-infected and non-infected ⁵¹Cr-labeled mastocytoma P-815-X2 cells and L-929 fibroblasts, which were used as targets. Cytotoxic lymphocytes could be isolated from the mice as early as 2 days after infection with vaccinia virus. The highest cytotoxic effect was obtained with lymphocytes taken 6 days after infection. The degree of lysi was correlated with the ratio of immune lymphocytes to target cells. Specific blocking of target cell lysis resulted after addition of anti-vaccinia antibody from different sources. The effector cells could be characterized as T cells by elimination of macrophages and B cells. Target cell killing was only possible in a syngeneic system; allogeneic infected target cells were not lysed significantly.     

Killing of Legionella pneumophila by nitric oxide in gamma-interferon-activated macrophages.

The role of nitric oxide (NO) radicals in killing the intracellular bacterial pathogen Legionella pneumophila (Lp) was examined in infected macrophages. Murine (RAW 264.7) and human (HL-60) cell monolayers were treated with 100 U/ml gamma-interferon (IFN) and cocultured with Lp in the presence and absence of NGMMA, a specific inhibitor of NO production. Viable Lp in IFN-treated RAW 264.7 cells decreased from 3.8 to 0.7 +/- 0.12 log CFU/ml after 24 h incubation, whereas in IFN+NGMMA-treated RAW 264.7 cells, viable Lp persisted at 2.2 +/- 0.2 log CFU/ml after 24 h. This increased survival corresponded with an inhibition of NO production (5.65 +/- 2.99 microM with NGMMA vs. 58.6 +/- 5.36 microM without NGMMA). Viable Lp were susceptible to killing, in a dose-dependent fashion, by 0, 2.5, and 5.0 mM sodium nitroprusside, a source of NO radicals. IFN-treated RAW 264.7 cells also had significantly decreased levels of intracellular iron (below assay limit) when compared to IFN+NGMMA-treated cells (72.0 +/- 0.78% of control). Normally permissive HL-60 cells treated with IFN were bacteriostatic rather than bactericidal, and NO production was not detected above background. Thus, NO radicals play a critical role in the bactericidal activity against Lp by IFN-treated RAW 264.7 cells, but the absence of NO production limits IFN-treated HL-60 cells to bacteriostasis.     

Author index

CBA spleen cells have been stimulated in vitro with A/Jap influenza virus-infected CBA spleen cells to generate a ‘primary’ cytotoxic lymphocyte (CL) response. The culture conditions were devised to allow the segregation of individual clones of CL and cytotoxicity measured by the lysis of infected or non-infected L-929 cells. The specificity was assessed by splitting clones and measuring the ability of the clones to discriminate between pairs of targets. Influenza A/FMI and A/Jap strains were used. Subsets of clones were detected which could lyze either A/Jap-infected or A/FMI-infected target cells. In addition CL clones were found which lyzed uninfected L-929 cells and a fourth category were clones which could not discriminate between A/FMI- and A/Jap-infected targets.     

Guinea pig kurloff (NK-like) cells mediate TNF-dependent cytotoxic activity: Analogy with NC effector cells

Kurloff cells are mononuclear cells possessing a large cytoplasmic inclusion body specific to the guinea pig. In this report, we present strong evidence that Kurloff cells can mediate NC activity against tumor cells in addition to their previously reported NK activity. Using an 18 h⁵¹Cr-release assay we have shown that Kurloff cells were highly effective in killing the TNF-sensitive WEHI 164 target cell line. Lower but significant cytotoxic activity was also observed after only 4 h. However, our results suggest a different mechanism of lysis in the 4 h and 18 h assay. Lysis of WEHI 164 target cells by Kurloff cells in the 4 h assay could be strongly increased in the presence of TPA alone or in combination with ionomycin whereas ionomycin alone was uneffective. In contrast, stimulation of Kurloff cells for 18 h with ionomycin alone or in combination with TPA could induce the release of TNF-like factor(s) as observed by the TNF bioassay using L-929 TNF-sensitive target cells. Release of TNF-like factor(s) could also be induced by stimulation with WEHI 164 target cells. Supernatants of Kurloff cells stimulated for 18 h with TPA + ionomycin were also highly cytotoxic against WEHI 164 target cells, but not against the TNF-resistant P815 target cell line. Pretreatment of these supernatants with antimurine TNF antibodies could almost completely inhibit their cytotoxic activity against WEHI 164 target cells. In contrast, supernatants of Kurloff cells stimulated for only 4 h did not show any TNF-like activity against the L-929 target cell line and were not cytotoxic against WEHI 164 target cells even after 18 h. Taken together, these results suggest that Kurloff cells can mediate NC activity against tumor cells in addition to their previously reported NK activity. By using multiple lytic pathways, these cells may play a crucial role in anti-tumor surveillance and defenses.Supported by grants from the National Cancer Institute and the Medical Research Council of Canada.Recipient of a studentship from the F.C.A.R.     

Difference in sensitivity to interferon among mouse hepatitis viruses with high and low virulence for mice

Mouse hepatitis viruses (MHV) of different virulence for mice were studied with respect to interferon (IFN) sensitivity. The growth of low-virulent MHV-S and intermediately virulent MHV-JHM was significantly suppressed in IFN-treated L cells compared with untreated cells. However, a comparable suppression of the growth of highly virulent MHV-2 was not observed in IFN-treated cells. This differential effect of IFN treatment could also be demonstrated at the level of viral mRNA and viral proteins. In cells infected with MHV-S or MHV-JHM the amount of viral mRNAs was remarkably reduced by IFN treatment. Also the levels of the major intracellular viral proteins, in particular the E1 protein, were affected by IFN treatment. Similar effects could not be demonstrated in MHV-2-infected cells. These results suggest that during MHV-S or MHV-JHM infection IFN treatment suppresses virus replication at several stages. The significance of these results is discussed in terms of the pathogenecity of these viruses.     

Effect of mosquito salivary gland treatment on vesicular stomatitis New Jersey virus replication and interferon alpha/beta expression in vitro

The sensitivity of vesicular stomatitis (VS) viruses to interferon (IFN)-mediated antiviral effects has been well documented. Previous studies in our laboratory have shown the ability of mosquito saliva to enhance vesicular stomatitis New Jersey (VSNJ) virus infection in mice. To investigate the effect of mosquito saliva on virus replication and IFN alpha/beta expression, virus titers were analyzed at various time points after infection in cells that were treated with mosquito salivary gland homogenate (SGH). Salivary gland treatment of mouse fibroblast cells (L929) resulted in a significant increase in virus growth kinetics compared with untreated controls. In contrast, Vero cells, which are deficient in the IFN alpha/beta response, did not yield increased viral titers in the time points examined. Treatment of L929 cells with an IFN alpha/beta neutralizing antibody also slightly increased virus yield. Ribonuclease protection assays revealed that induction of IFN alpha2 expression was reduced in L929 cells treated with SGH. Modulation of IFN alpha/beta by mosquito saliva may be a critical determinant of the transmission and pathogenesis of VSNJ virus.     

A comparison between two methods for measuring tumor necrosis factor in biological fluids

The current study was undertaken to compare two methods for the efficiency of measuring tumor necrosis factor (TNF-α) in biological fluids, which is species undependent, reliable, sensitive, simple and not expensive. We have compared the MTT tetrazolium cytotoxic assay [1,2] and the³H-thymidine (³H-TdR) incorporation cytostatic assay for measuring the anti-tumor activity of human recombinant TNF-α, of human colonic tissue and of supernatants ofin vitro stimulated human and rat peritoneal macrophages. Two target cell-lines, namely murine myelomonocytic leukaemia WEHI-164- and L-929-transformed murine fibroblast cell-lines, were used in the MTT assay. The L-929 line was also used in the³H-TdR assay. WEHI-164 was more sensitive than the L-929 cell-line in the MTT cytotoxic assay. Furthermore, the MTT assay was more sensitive to TNF-α than the³H-TdR assay. Both methods can be used for the detection of anti-tumor activity in biological fluids but the MTT cytotoxic method has the advantage of being more sensitive and more simple.

     

Effects of synthetic bis-β-chloroethylamine estrogen derivatives on proliferation of skin sarcoma cells

The effects of four new synthetic bis-β-chloroethylamine-containing estrogens and known cytostatic agents chlorophenacyl and estradiol mustard were compared on monolayer cultures of transformed L-929 fibroblasts (from murine skin sarcoma). The drugs within the concentration range of 10⁻⁵-5×10⁻⁷M inhibited proliferation of cultured cells by 67%. Chlorophenacyl displayed the least antiproliferative activity (15% inhibition at 10⁻⁵M). Steroid nucleus introduced into the molecule enhanced antiproliferative activity of test drug in comparison with chlorophenacyl, probably due to accumulation of the hormone-cytostatic molecules in cells. Estradiol had no effect on proliferative activity of L-929 cells, and no specific estrogen-binding sites were found in cultured transformed fibroblasts. The antiproliferative effect of hormone-cytostatics on this culture is not mediated via specific interactions with estrogen receptors. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 129, No. 6, pp. 695–697, June, 2000