Interferon-gamma enhances target cell sensitivity to monocyte killing

The mechanism of human peripheral blood monocyte-mediated cytotoxicity was investigated using the HT-29 human colon adenocarcinoma line, A673 human rhabdomyosarcoma line, and A375 human melanoma line as target cells. Pretreatment of these target cells with 100 U/ml of recombinant human interferon (IFN)-gamma for 48 hr increased their susceptibility to monocyte killing. Increased susceptibility to the lytic action was particularly pronounced at low effector/target cell ratios. Unlike IFN-gamma human IFN-alpha did not potentiate monocyte cytotoxicity, and pretreatment of HT-29 with IFN-alpha also had virtually no effect on their susceptibility to monocyte killing. However, IFN-gamma appeared to prime either monocytes or target cells to become responsive to IFN-alpha. Our data suggest that IFN-gamma can promote the killing of tumor cells by monocytes through two separate actions, one on the monocyte and one on the target cell.     

Inhibition of mast cell-mediated cytotoxicity by IFN-alpha/beta and -gamma.

Although IFN enhance the cytotoxic activity of NK cells, K cells, and monocytes, IFN-alpha/beta and IFN-gamma did not stimulate the cytotoxic activity of rat peritoneal mast cells (PMC), but had an inhibitory effect. Preincubation for 2 h with 100 and 200 U/ml of IFN-gamma and IFN-alpha/beta, respectively, inhibited PMC cytotoxicity against WEHI-164 target cells. Lower concentrations of IFN-gamma (12.5 U/ml) and IFN-alpha/beta (25 U/ml) inhibited cytotoxicity of PMC after 8 h preincubation. The inhibitory effect of IFN was concentration and time dependent. In contrast to cytotoxicity, the release of histamine by PMC was not stimulated by the target cells WEHI-164 and there was no correlation between histamine release and cytotoxic activity of PMC. Specific antibody to subclasses of IFN prevented the inhibition of PMC cytotoxic activity, but preincubation with antibodies to the alternate subclass of IFN did not affect the observed inhibition. Moreover, the presence of both subclasses of IFN showed an additive inhibition of PMC cytotoxicity. The cytotoxic activity of PMC can be completely inhibited by the addition of anti-TNF during the assay. At high concentrations (400 U/ml), IFN inhibited the release of TNF from PMC. In the presence of RNA or protein synthesis inhibitors, IFN did not inhibit cytotoxicity of PMC further. We postulate that IFN may alter gene expression in mast cells in a manner that down-regulates their functions.     

Virus-induced interferon alpha/beta (IFN-alpha/beta) production by T cells and by Th1 and Th2 helper T cell clones: a study of the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta on functions of different T cell populations

Spleen cells, resting T cells, activated T cells, and T cell clones characterized as type 1 (Th1) and type 2 (Th2) were investigated for their ability to produce interferon (IFN) following in vitro culture with Newcastle disease virus (NDV). All of the above cell populations, including both Th1 and Th2 T cell clones, produced high levels of IFN following in vitro culture with NDV. This IFN was characterized as a mixture of IFN-alpha and IFN-beta with IFN-alpha being the predominate species of IFN contained in the mixture. IL-2 greatly enhanced the production of IFN-alpha/beta by all cell populations in response to NDV. These different T cell populations responded very differently to the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta. IFN-alpha/beta was shown to be a potent inhibitor of Con A or IL-2-induced proliferation of different T cell populations. This inhibition was not associated with a reduction in lymphokine production since spleen cells or Th1 T cell clones cultured with Con A and IFN-alpha/beta had no decrease in IL-2 or IFN-gamma production when compared to Con A-stimulated control cultures. IFN-gamma had little to no inhibitory activity on Con A-induced proliferation of spleen cells. In fact, Con A-induced proliferation was usually enhanced by IFN-gamma when nylon wool-enriched T cells were assessed. Different results were observed when IFN-gamma and IFN-alpha/beta were investigated for their ability to inhibit IL-2-induced proliferation of different T helper cell clones. IFN-gamma and IFN-alpha/beta were both capable of inhibiting IL-2-induced proliferation of T cell clones characterized as type 2 (Th2). In contrast, IFN-gamma had no effect on IL-2-induced proliferation of Th1 clones. IFN-alpha/beta, however, inhibited IL-2-induced proliferative responses of both Th1 and Th2 T cell clones. These results document the facts that (1) IFN-gamma and IFN-alpha/beta differ in their immunoregulatory actions, (2) different T cell subpopulations vary in their susceptibility to IFN-gamma regulation, and (3) virus induction of IFN-alpha/beta appears to be a ubiquitous function associated with different T cell populations.     

Interferon-gamma is a strong modulator of NK susceptibility and expression of beta 2-microglobulin but not of transferrin receptors of K562 cells

The human cell line K562 was treated with human natural leukocyte interferon (IFN-alpha) and recombinant immune interferon (IFN-gamma). Cell cultures exposed to both types of IFNs displayed a reduced susceptibility to the cytotoxic activity of human PBL (NK activity). While this effect occurred preferentially at high doses of IFN-alpha, as little as 10 U/ml of IFN-gamma caused a marked decrease in susceptibility to NK-cell-mediated lysis. Using a monoclonal antibody against human beta2-microglobulin (beta2M) a low level of specific binding to K562 cells was detected. The binding increased after treatment with IFN-alpha (1.4-fold) and IFN-gamma (1.7-fold). The expression of transferrin receptors (TR) was not changed significantly. A hybrid cell line between K562 and a Burkitt's lymphoma-derived cell line displayed a similar pattern of response to IFN-alpha and IFN-gamma as did K562, when effects on NK susceptibility, beta2M expression, and TR expression were studied. The Burkitt's lymphoma line PUT showed no consistent changes in expression of beta2M and TR. These results demonstrate that IFN-gamma is highly efficient in modulating the NK susceptibility, and the expression of beta2M on K562. The presented data do not support a role for expression of TR as the only property that determines the degree of NK susceptibility, since there was no correlation between NK susceptibility and TR expression among the cell lines tested or when IFN-treated and untreated cells were compared.     

Interferon-induced resistance to the killing by NK cells: a preferential effect of IFN-gamma

HeLa cells show a decrease of susceptibility to the killing by natural killer (NK) cells when treated with IFN-alpha, beta, or gamma. The concentrations at which preparations of IFN-alpha or beta induce the resistance to killing are those which also induce resistance of HeLa cells to infection by vesicular stomatitis virus (VSV). Stimulation of the killing activity of NK cells is also induced at that same range of concentrations of IFN-alpha and beta. In contrast with preparations of IFN-gamma, induction of the resistance to killing occurs at IFN concentrations which have only marginal stimulatory effect on the activity of NK cells and have no antiviral effect against VSV. IFN-gamma, produced with cloned IFN-gamma cDNA, is as effective as lymphocyte-produced IFN in inducing the resistance to natural killing. The potent effect of IFN-gamma on the target cells is, therefore, not due to the function of lymphokines which might contaminate lymphocyte-produced preparations of IFN-gamma, but a genuine property of the IFN itself.     

The effect of stress on interferon production in mice

In order to study the effect of stress on the interferon (IFN) production, we determined the IFN-alpha/beta and -gamma producing capacities in restraint-stressed and SART-stressed mice. Restraint-stress caused not only a significant reduction in body weight, but also a reduction in the weight of spleen. The IFN-alpha/beta producing capacity by Newcastle disease virus (NDV) was significantly lower in the stressed mice. The IFN-gamma producing capacity by both Con-A and PHA-P was also depressed by this stress. On the other hand, SART-stress, whose severity was thought to be mild because no loss of body and spleen weights was seen, did not affect IFN-alpha/beta and -gamma producing capacities. These results suggest that the suppression of IFN producing capacity is dependent on the severity of physical stress.     

Synergistic antiviral and antiproliferative activities of Escherichia coli-derived human alpha, beta, and gamma interferons

The antiviral and antiproliferative effects of highly purified Escherichia coli-derived human interferons (IFNs) were examined in human melanoma cells (Hs294T). Antiproliferative activity was monitored by measuring inhibition of cell multiplication, and antiviral activity was determined by inhibition of herpes simplex virus type 1 replication. Treatment of cells with IFN-gamma in combination with IFN-alpha A or IFN-beta 1 resulted in potentiation of both antiproliferative and antiviral activities. In contrast, combination treatments composed of IFN-alpha A and IFN beta 1 yielded inconsistent results. Some combinations reflected additive responses, whereas others were antagonistic. To examine correlations between IFN-induced biological activities and interactions of the different IFNs with cell surface receptors, in vivo [35S]methionine-labeled IFN-alpha A was prepared. Binding studies indicated the presence of 2,980 +/- 170 receptors per cell, each with an apparent Kd of (8.4 +/- 1.3) X 10(-11) M. Results from competitive binding studies suggested that Hs294T cells possess at least two types of IFN receptors: one which binds IFN-alpha A and IFN-beta 1 and another to which IFN-gamma binds.     

IFN-gamma treatment of K562 cells inhibits natural killer cell triggering and decreases the susceptibility to lysis by cytoplasmic granules from large granular lymphocytes

Pretreatment of human K562 leukemia cells with rIFN-alpha and rIFN-gamma resulted in decreased susceptibility to lysis by human peripheral blood NK cells. The reduction of NK-susceptibility after IFN treatment was not due to a general effect of IFN on the stability of the cell membrane because the susceptibility of K562 cells to lysis by antibodies plus C, distilled water, or lysolecithin was unaffected. Binding studies with effector cell preparations enriched for NK cells with large granular lymphocyte morphology revealed no difference in binding to control and IFN-gamma-treated target cells. The sensitivity to soluble NK cytotoxic factors was not affected significantly by the IFN treatment. In contrast, the susceptibility of IFN-treated target cells to the cytotoxic activity of purified cytoplasmic granules from a rat large granular lymphocyte tumor was significantly reduced, indicating that the IFN-induced resistance acted at the level of susceptibility to the lytic mechanism of NK cells. However, IFN-alpha was more effective than IFN-gamma in inducing resistance to the cytoplasmic granules although resulting in only a weak resistance in the cell-mediated cytotoxic assay. IFN-gamma but not IFN-alpha caused a reduction in the frequency of effector cells that had reoriented their Golgi apparatus toward their bound target cell. In addition, IFN-gamma treated K562 cells failed to elicit an influx of Ca2+ into effector cells. Taken together, the results suggest that IFN-gamma in addition to an increased resistance to the lytic molecules released by NK cells can also induce changes in the target cells which prevent the triggering and activation of the effector cell.     

The vaccinia virus soluble alpha/beta interferon (IFN) receptor binds to the cell surface and protects cells from the antiviral effects of IFN

Poxviruses encode a broad range of proteins that interfere with host immune functions, such as soluble versions of receptors for the cytokines tumor necrosis factor, interleukin-1 beta, gamma interferon (IFN-gamma), IFN-alpha/beta, and chemokines. These virus-encoded cytokine receptors have a profound effect on virus pathogenesis and enable the study of the role of cytokines in virus infections. The vaccinia virus (VV) Western Reserve gene B18R encodes a secreted protein with 3 immunoglobulin domains that functions as a soluble receptor for IFN-alpha/beta. We have found that after secretion B18R binds to both uninfected and infected cells. The B18R protein present at the cell surface maintains the properties of the soluble receptor, binding IFN-alpha/beta with high affinity and with broad species specificity, and protects cells from the antiviral state induced by IFN-alpha/beta. VV strain Wyeth expressed a truncated B18R protein lacking the C-terminal immunoglobulin domain. This protein binds IFN with lower affinity and retains its ability to bind to cells, indicating that the C-terminal region of B18R contributes to IFN binding. The replication of a VV B18R deletion mutant in tissue culture was restricted in the presence of IFN-alpha, whereas the wild-type virus replicated normally. Binding of soluble recombinant B18R to cells protected the cultures from IFN and allowed VV replication. This represents a novel strategy of virus immune evasion in which secreted IFN-alpha/beta receptors not only bind the soluble cytokine but also bind to uninfected cells and protect them from the antiviral effects of IFN-alpha/beta, maintaining the cells' susceptibility to virus infections. The adaptation of this soluble receptor to block IFN-alpha/beta activity locally will help VV to replicate in the host and spread in tissues. This emphasizes the importance of local effects of IFN-alpha/beta against virus infections.     

Alternative induction of alpha/beta interferons and gamma interferon by listeria monocytogenes in mouse spleen cell cultures

Production of interferon (IFN) by Listeria monocytogenes (LM) in nonimmunized mouse spleen cell cultures was studied. IFN-gamma defined by virtue of its acid stability and antigenicity was produced in spleen cell cultures obtained from ddY mice, C57BL/6 mice, and BALB/c mice in response to heat-killed (HK) LM within 24 hr. On the other hand, production of IFN-alpha/beta was demonstrated in spleen cell cultures obtained from one of four nude mice (BALB/c, nu/nu). Therefore, it is important to know the reason why the spleen cells of mice other than nude mice did produce only IFN-gamma, but did not produce IFN-alpha/beta in response to HK-LM. Spleen cells obtained from ddY mice were fractionated, and the cellular source for IFN production of either IFN-alpha/beta or IFN-gamma induced by HK-LM was investigated. IFN-gamma was produced only by a mixture of T lymphocytes (nylon wool-nonadherent, Thy-1-positive cells) and macrophages by HK-LM. Neither T lymphocytes nor macrophages alone produced IFN by HK-LM. Macrophage-depleted spleen cells produced neither IFN-gamma nor IFN-alpha/beta, but these cells acquired the ability to produce IFN-alpha/beta, not IFN-gamma, only when they had been treated with IFN-alpha/beta. A possible mechanism of both IFN-gamma and IFN-alpha/beta induction by Listeria in mouse spleen cell cultures is discussed.     

Interferon-gamma reduces macrophage-suppressive activity by inhibiting prostaglandin E2 release and inducing interleukin 1 production

Normal peritoneal M phi of C3H/HeN mice were able to suppress lymphocyte proliferation in a dose-dependent fashion when added to Con A-pulsed spleen cell cultures. However, M phi-suppressive activity could be partially or completely reduced by in vitro pre-exposure to nonimmune IFN-alpha or immune recombinant IFN-gamma. For both IFN-alpha and IFN-gamma, reduction of M phi suppression was marginal at 10(1) U/ml and became highly significant at 10(2) to 10(3)/ml. The ability of IFN-alpha and IFN-gamma to modulate M phi suppression appears to be related to distinct mechanisms. In fact, impairment of M phi suppression by IFN-alpha occurred in parallel to the decrease of M phi capacity to produce PGE2 and the oxygen intermediate O2-, two molecules responsible for M phi-suppressive activity. In contrast, M phi exposed to IFN-gamma showed only impairment of PGE2 production, whereas O2- release was not significantly affected. Furthermore, at variance with IFN-alpha, IFN-gamma directly stimulated M phi to synthesize and release IL 1, a monokine known to promote lymphocyte proliferation.     

Introduction of the H-2Dk gene into a class I-negative tumor cell line confers interferon-gamma inducibility upon the silent endogenous H-2Kk gene.

Kgv cells do not constitutively express class I mRNA or protein. Interferon (IFN)-gamma, but not IFN-alpha/beta, induces H-2Dk expression. IFN does not induce H-2Kk expression. We examined constitutive and IFN-inducible class I expression on Kgv cells stably transfected with genomic clones of H-2Kk or H-2Dk and on somatic cell hybrid lines constructed between Kgv cells and constitutively class I-positive cells of a distinguishable H-2 haplotype. Our results suggest that both the lack of constitutive class I expression and the inability of IFN-alpha/beta to induce class I expression on Kgv cells are primarily due to cis-regulatory mechanisms. However, stable introduction of the H-2Dk gene into Kgv cells conferred IFN-gamma inducibility upon the silent endogenous H-2Kk gene. Therefore, the failure of IFN-gamma to induce H-2Kk expression on Kgv cells is due, at least in part, to a trans-regulatory mechanism.     

Alterations of interferon production in a mouse model of thermal injury

The effect of thermal injury on the response of interferon (IFN) production in vivo and in vitro after stimulation with eight representative inducers was investigated in a mouse model. The response of mice to immune IFN (IFN-gamma) inducers, staphylococcal enterotoxin A, concanavalin A, and a specific antigen for BCG-sensitized lymphocytes (purified protein derivative) was impaired after a 30% total body surface area third-degree burn. Suppression of IFN-gamma production was observed at day 2 and persisted until day 7 after burn. Decreased IFN-gamma production correlated closely with the percentage of total body surface area burned. When virus type IFN (IFN-alpha/beta) inducers, Newcastle disease virus, polyriboinosinic-polyribocytidylic acid, 10-carboxymethyl-9-acridanone, and E. coli endotoxin, were administered to mice, no change in IFN response was observed after thermal injury. Similar results were obtained when spleen cells obtained from thermally injured mice were stimulated with IFN-gamma inducers in vitro. These studies suggest that although the capacity for IFN-alpha/beta production remains intact in thermally injured mice, IFN-gamma production may be selectively decreased in burned animals and in their spleen cells.     

Mutant cell lines unresponsive to alpha/beta and gamma interferon are defective in tyrosine phosphorylation of ISGF-3 alpha components.

The 84-, 91-, and 113-kDa proteins of the ISGF-3 alpha complex are phosphorylated on tyrosine residues upon alpha interferon (IFN-alpha) treatment and subsequently translocate to the nucleus together with a 48-kDa subunit. In this study, we investigated the presence and the functional status of ISGF-3 alpha subunits and Tyk-2 and JAK1 tyrosine kinases in mutant HeLa cells defective in the IFN-alpha/beta and -gamma response. Stable cell fusion analysis revealed a single complementation group among one class (class B) of mutants. The class B mutants contain detectable level of mRNA and proteins of the 84-, 91-, and 113-kDa proteins, but neither the protein nor mRNA is inducible by IFN-alpha or -gamma. The 91-kDa protein IFN-gamma-activated factor fails to be activated into a DNA-binding state after IFN-alpha or -gamma treatment. In addition, the 91-kDa protein is unable to localize in the nucleus after IFN-alpha and -gamma treatment, and the 113-kDa protein fails to translocate after IFN-alpha treatment. Immunoprecipitation studies document a failure of phosphorylation of the 84- or 91-kDa proteins after IFN-alpha or -gamma treatment. Similarly, no tyrosine-phosphorylated 113-kDa protein was detected after IFN-alpha treatment. The inability of class B mutants to phosphorylate the 84-, 91-, or 113-kDa protein on tyrosine residues correlated with the loss of biological response to IFN-alpha and -gamma. The genetic defect appears to be the absence of the tyrosine kinase JAK1. Our data therefore confirm a recent report that JAK1 plays a critical early signaling role for both IFN-alpha/beta and IFN-gamma systems.     

Recombinant interferon-alpha, -beta, and -gamma enhance the proliferative response of human B cells

Recombinant interferons (IFN-alpha, -beta, and -gamma) were examined for their effects on B cell activation. Relatively small IgM+ B cells from human blood samples were isolated by fluorescence-activated cell sorting and were used as target cells. Although the interferons themselves were nonmitogenic, each enhanced the proliferative response induced by a mitogenic anti-mu monoclonal antibody, with IFN-beta usually showing the greatest enhancement and IFN-gamma the least. Pretreatment with the interferons primed resting B cells to undergo enhanced DNA synthesis in response to the anti-mu antibody DA4. Conversely, anti-mu pretreatment, followed by IFN treatment, did not induce B cells to enter the S phase. Time-course analysis revealed that IFN could augment the anti-mu response even when added as late as the final 24 hr of a 3-day culture interval. Combinations of IFN-gamma plus IFN-alpha or -beta were synergistic in the anti-mu response, whereas the IFN-alpha plus IFN-beta combination was not. The data suggest that interferons produced by both lymphocytes (IFN-gamma) and nonlymphoid inflammatory cells (IFN-alpha and -beta) can enhance B cell growth via different mechanisms.     

Induction of receptors for tumor necrosis factor-alpha by interferons is not a major mechanism for their synergistic cytotoxic response

We have investigated the effects of various interferons on the receptors for recombinant tumor necrosis factor-alpha (rTNF-alpha) and also their effects on rTNF-alpha-mediated cytotoxicity on human cervical carcinoma cell line ME-180. Preincubation of cells with interferon (IFN)-gamma causes a concentration- and time-dependent increase in rTNF-alpha receptor number without any change in the affinity constant of the receptors. The increase in receptor number is caused only by IFN-gamma and not by IFN-alpha or IFN-beta. Approximately 4-6 h of preincubation with IFN-gamma are required for maximum increase in rTNF-alpha binding to the cells, and this increase can be abolished by inhibitors of protein synthesis, suggesting de novo synthesis of rTNF-alpha receptors. The half-life of both uninduced and induced receptors of rTNF-alpha is approximately 2 h, indicating a rapid turnover. The binding of rTNF-alpha to the cells can also be eliminated by pretreatment of cells with trypsin. Following the removal of trypsin, binding of rTNF-alpha gradually increases, and this requires the synthesis of new proteins. The cytotoxic effect of rTNF-alpha on ME-180 cells is potentiated severalfold by the addition of either IFN-alpha, -beta, or -gamma. However, at similar concentrations, relatively higher potentiation of rTNF-alpha cytotoxicity is observed with IFN-gamma as compared to IFN-alpha and IFN-beta. The pre-exposure of cells to IFNs is as effective as co-exposure in enhancing cytotoxic effects of TNF-alpha. The induction of TNF-alpha receptors by IFNs is observed in different cell types regardless of their sensitivity to TNF-alpha, suggesting that increase in receptor number alone is not sufficient for the enhanced cytotoxic response. Because the enhancement of cytotoxic effects of TNF-alpha is observed by all IFNs but receptor induction in ME-180 cells occurs only with INF-gamma and because metabolic inhibitors which down-regulate TNF-alpha receptors also enhance cytotoxic response, we suggest that the induction of TNF-alpha receptor by IFNs is not a major mechanism of synergism between these cytokines.