T cell regulation of interferon-alpha/beta (IFN-alpha/beta) production by alloantigen-stimulated bone marrow cells

We have previously reported that mouse bone marrow cells produce high levels of interferon-alpha/beta (IFN-alpha/beta) after 5 to 6 days of in vitro culture with irradiated allogenic spleen cells. The current study was initiated to determine whether or not T cells are important for alloantigen-induced IFN-alpha/beta production by mouse bone marrow cells. Bone marrow cells and spleen cells were obtained from C57BL/6 mice. These cells were treated with different monoclonal antisera and complement, and then were cultured 5 to 6 days with irradiated DBA spleen cells. The results from these experiments indicated that optimal IFN-alpha/beta production by alloantigen-stimulated bone marrow cells required Lyt-1+2+ T cells. In addition, when bone marrow cells obtained from nu/nu B10 mice were cultured with alloantigen, only low levels of IFN were produced when compared with IFN production by bone marrow cells obtained from normal littermate B10 mice. The addition of nylon wool-enriched splenic T cells to cultures containing bone marrow cells and alloantigen resulted in an augmentation of IFN-alpha/beta production by three-fold to fivefold. Furthermore, bone marrow cells obtained from alloantigen-immunized mice produced much higher levels of IFN-alpha/beta and in a shorter period of time (2 to 3 days) when compared with bone marrow cells obtained from control or non-immunized mice. Cyclosporin A (CsA) has been shown to inhibit predominantly T cell-dependent responses. The effect of CsA on IFN production by alloantigen-stimulated bone marrow and spleen cells was investigated. The addition of CsA at concentrations as low as 0.1 micrograms/ml inhibited not only IFN-gamma production by alloantigen-stimulated spleen cells, but also IFN-alpha/beta production by alloantigen-stimulated bone marrow cells. In contrast, IFN-alpha/beta production by Newcastle disease virus-infected spleen cells, bone marrow cells, or L cells was not inhibited by the addition of CsA (1 microgram/ml). Thus, the ability of bone marrow cells to produce high levels of IFN-alpha/beta after in vitro culture with alloantigen is dependent upon T cells resident in the bone marrow. IFN-alpha/beta production by alloantigen-stimulated bone marrow cells may play a major role in the pathogenesis associated with graft-vs-host disease and in T cell regulation of hematopoiesis.     

The significance of alpha/beta interferons and gamma interferon produced in mice infected with Listeria monocytogenes

Interferon (IFN)-alpha/beta was induced in the circulation of mice infected intravenously with Listeria monocytogenes 24 to 72 hr after infection, but was not induced by the administration of heat-killed Listeria, listerial cell wall fraction (LCWF), or listerial soluble fraction. Appearance of IFN-alpha/beta showed a pattern similar to that of the growth of bacteria in the spleen and the liver of mice. IFN-alpha/beta production was abrogated by pretreatment of mice with anti-asialo GM1 antibody, antithymocyte serum, or hydrocortisone, but not with cyclophosphamide or carrageenan. Such treatments which suppressed IFN-alpha/beta production did not influence bacterial growth in the organs of mice in the early stage of Listeria infection. Administration of IFN-alpha/beta exogenously also did not. After 5 days of infection when the specific resistance against reinfection with Listeria was established, IFN-gamma but not IFN-alpha/beta was induced in the circulation 3 to 6 hr after stimulation with LCWF or reinfection with Listeria. IFN-gamma production was abrogated completely by cyclophosphamide and antithymocyte serum, and partially by hydrocortisone and carrageenan, but not by anti-asialo GM1 antibody in Listeria-infected mice treated with these agents before induction of IFN-gamma by LCWF. Presumably, IFN-alpha/beta might be produced by asialo GM1-bearing cells but IFN-gamma might not. However, IFN-gamma production was suppressed in Listeria-infected mice, when IFN-alpha/beta production had been inhibited by treatment with anti-asialo GM1 antibody or when the IFN produced had been neutralized with anti-mouse IFN-alpha/beta antibody. Therefore, it is conceivable that IFN-alpha/beta might be essential for the generation or the expression of antigen-specific T cells involving IFN-gamma production and acquired resistance during Listeria infection. In fact, the bacterial growth in the organs of mice in the early stage of infection was normal in IFN-alpha/beta-depleted mice but it resulted in the delay of T-cell-dependent elimination of bacteria from the organs of mice in the late stage.     

Enhanced interferon-alpha/beta (IFN-alpha/beta) and defective IFN-gamma production in chronic graft versus host disease: a potential mechanism for immunosuppression

Immunosuppression is a well-characterized consequence of chronic graft-versus-host disease (GVHD). We have previously shown that interferon (IFN) is produced in high levels during acute GVHD. Our objective in this study was to determine if IFN, as a cytokine with known immunosuppressive qualities, could be detected in mice experiencing chronic GVHD-induced immunosuppression. Two different experimental models were used to induce chronic GVHD. The first model involved the injection of parental strain spleen cells into adult F1 hybrids (AJ----B6AF1), while the second model utilized GVHD induced across minor histocompatibility barriers (B10.D2----BALB/c). Results indicated that significant levels of serum IFN-alpha/beta are present in mice undergoing chronic GVHD. Spleen cells from chronic GVHD mice were also shown to produce significant levels of IFN-alpha/beta upon in vitro culture in medium only. This IFN-alpha/beta production was greatly increased when GVHD spleen cells were cultured with either concanavalin A (Con A) or IL-2. In contrast, IFN-gamma production was undetectable in these Con A- or IL-2-containing cultures. Additionally, these same spleen cells which produced high levels of IFN-alpha/beta were immunosuppressed as measured by mitogen-induced cell proliferation. These results suggest that IFN-gamma production is defective in GVHD spleen cells, and that the presence of high IFN-alpha/beta production by GVHD mice may contribute to the immunosuppression associated with chronic GVHD.     

Interferon-gamma induction by lipopolysaccharide: dependence on interleukin 2 and macrophages

Bacterial lipopolysaccharide (LPS) induced fresh murine splenocytes to produce interferon (IFN)-alpha/beta presumably by stimulation of the B lymphocytes and macrophages. However, when the splenocytes were "aged" for 24 to 72 hr in culture before addition of the LPS, the IFN response was significantly increased and was determined to be predominantly IFN-gamma. Because low levels of interleukin 2 (IL 2) were found to be spontaneously produced by the unstimulated splenocytes during the "aging" process, the effect of IL 2 on IFN induction by LPS in fresh splenocytes was examined. The addition of LPS to freshly prepared splenocyte cultures that were treated with human IL 2, either native or recombinant, before exposure to the LPS resulted in the LPS inducing large amounts of IFN-gamma. IL 2 alone induced little if any IFN in the splenocyte cultures. Depletion of T cells and large granular lymphocytes (LGL) from the cultures by anti-Thy-1.2 antibodies plus complement abrogated IFN-gamma production, and the addition of polymyxin B to "aged" splenocyte cultures resulted in loss of IFN production in response to LPS. Cultures that were enriched for T cells and LGL by passage through nylon wool produced significant amounts of IFN-gamma in response to LPS only if first treated with IL 2. Furthermore, the addition of splenic adherent cells to purified nylon wool-non-adherent (NWNA) cells augmented IFN-gamma production, whether or not the NWNA cells were pretreated with IL 2. This enhancement appeared to require direct contact between adherent cells and NWNA cells, because physical separation abrogated IFN production. The addition of recombinant IL 1 or LPS-conditioned supernatants of macrophage cultures did not replace adherent cell activity. These data demonstrate that LPS, which predominantly induces IFN-alpha/beta in fresh murine splenocytes, is able to stimulate T lymphocytes to produce IFN-gamma if the T cells are first exposed to endogenously produced or exogenously applied IL 2. Because IFN-gamma is a potent activator of the bactericidal and cytocidal potential of macrophages, the induction of IFN-gamma by bacterial LPS may play an important role in resistance/recovery mechanisms against bacterial infections.     

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.     

Sequential production of alpha and beta interferons and gamma interferon in the circulation of Listeria monocytogenes-infected mice after stimulation with bacterial lipopolysaccharide

Sequential production of interferon (IFN)-alpha/beta and IFN-gamma in the circulation of mice which had been previously infected with viable Listeria monocytogenes was induced by injection of lipopolysaccharide (LPS) derived from Salmonella typhimurium. IFN-alpha/beta production occurred 2 hr after injection of LPS, thereafter IFN-gamma appeared and the maximum titer was demonstrated at 6 hr. At that time, almost all of the IFN was IFN-gamma. IFN-gamma production in response to LPS was observed from the 5th through the 11th day after infection with Listeria, but it was not demonstrated in either mice infected with lower doses of viable Listeria or mice immunized with heat-killed bacteria. IFN-alpha/beta production was not drastically affected by treatment with hydrocortisone, cyclophosphamide, carrageenan, antithymocyte serum, or anti-asialo GM1 antibody, whereas IFN-gamma production was suppressed by administration of all those agents. Noteworthily, IFN-alpha/beta, but not IFN-gamma, was produced even 6 hr after stimulation with LPS in cyclophosphamide- or antithymocyte serum-treated mice. IFN-gamma induction by LPS was markedly suppressed in mice in which IFN-alpha/beta produced by Listeria infection itself had been depleted by treatment with anti-mouse IFN-alpha/beta antibody, but it was not inhibited in mice when IFN-alpha/beta induced not by Listeria infection but by LPS had been depleted by treatment with anti-mouse IFN-alpha/beta antibody.     

Selective inhibition of the generation of T suppressor cells of contact sensitivity in vitro by interferon

The T suppressor (Ts) cell response in contact sensitivity is preferentially inhibited by murine interferon-alpha, beta (IFN-alpha, beta) in vivo. Previous studies in vivo have suggested that IFN exerts its effect directly on the Ts subpopulation rather than through an effect on antigen-presenting macrophages. Nevertheless, the mechanism of this selective blockade remained unclear. To better define the mechanism(s) of inhibition of suppression by IFN-alpha, beta, we determined whether IFN acted on lymphocytes, macrophages, or both. Antigen-specific T effector cells of delayed-type hypersensitivity (TDH) and Ts cells were induced in vitro by co-culture of spleen lymphocytes with bone marrow-derived antigen-presenting macrophages (BM-MA) pulse-labeled with 2,4-dinitrobenzene sulfonate (DNBSO3). TDH or Ts activity was demonstrated by transfer of the lymphocytes into naive recipient BALB/c mice after 3 days of culture. BM-MA cultured for 5 to 7 days (BM-MA d5-7) before labeling preferentially activated TDH cells (Thy-1+, Lyt-1+2-); 10- to 14-day-old BM-MA (BM-MA d10) induced Ts cells (Thy-1+, Lyt-2+), as previously shown. Treatment of the spleen lymphocyte suspension with pure mouse IFN-alpha, beta at a dose of 10(3) U/10(8) cells completely blocked the induction of Ts cells but had no effect on the induction of TDH cells. Pretreatment of the antigen-presenting BM-MA for 24 hr with IFN (10(2) U/3 X 10(5) cells) had no effect on the induction of Ts and TDH cells. Cultivation of lymphocytes on a DNP-BM-MA d6 monolayer did not result in the induction of Ts cells; however, in the presence of a goat anti-murine IFN-alpha, beta antibody, Ts cells were induced. This finding indicates that the spontaneous release of IFN-alpha, beta in those cultures prevented the induction of Ts cells. These results confirm our previous observation that Ts cells are more easily blocked by IFN-alpha, beta than TDH cells, and demonstrate that IFN affects the Ts subpopulation not via modulation of the antigen-presenting macrophages. IFN-alpha, beta-producing, antigen-presenting, or accessory cells may therefore prevent the activation of this type of Ts cell.     

Modulation of IL 2-dependent growth of mouse NK cells by interferon and T lymphocytes

The regulatory role of interferon (IFN) on the growth of mouse natural killer (NK) cells in the presence of interleukin 2 (IL 2) was analyzed by the limiting dilution assay. Pretreatment for 5 hr with IFN (600 U/ml) was able to augment the frequency of proliferating cells and NK effector cells when spleen cells of BALB/c nu/+ and BALB/c nu/nu were cultured for 7 days in the presence of IL 2. When IFN was present during the 7-day culture period, we again found an increase in proliferative and cytotoxic frequencies in cultures of spleen cells from nude mice, but in contrast, found a decrease in these frequencies in cultures of spleen cells from euthymic mice. Addition of irradiated (3000 R) spleen or thymus feeder cells from euthymic mice to the nu/nu cultures caused an inhibitory activity of IFN also on nu/nu cells. These data indicate that IFN can have both positive and negative regulatory effects on the in vitro growth and differentiation of mouse NK cells and that the inhibitory effects are mediated via T lymphocytes.     

Inhibition of macrophage-induced antigen-specific T lymphocyte proliferation by poly I:C: strain and antigen independence

We have previously demonstrated that IFN-alpha/beta, poly I:C (an inducer of IFN-alpha/beta), and IFN-gamma can inhibit the ability of KLH-pulsed peritoneal macrophages to induce proliferation of syngeneic, KLH immune T lymphocytes in CBA/J mice. In this study, we show that this IFN-induced immunosuppression is not restricted to CBA/J (H-2k) mice but is also seen in BALB/cJ (H-2d) mice. A similar inhibition of proliferation is observed with the KLH-specific T cell hybridoma BDK, 100, which requires KLH-pulsed macrophages for optimum proliferation and IL-2 production. The immunosuppression produced by IFN was also independent of the antigen employed. Inhibition of T lymphocyte proliferation was observed when casein, instead of KLH, was used to immunize T cells and to pulse peritoneal macrophages in vivo. Utilizing KLH and casein, the antigen specificity of the inhibition was demonstrated. Therefore, the inhibition by the IFN-inducer poly I:C of macrophage-induced, antigen-specific T cell proliferation is not limited by H-2 type of the mice or to one antigen.     

Antibody response to simian virus 40 tumor antigen in nude mice reconstituted with T cells.

Athymic BALB/c nude mice (nu/nu) fail to generate circulating antibodies to simian virus 40 (SV40) tumor (T) antigen when immunized with SV40-transformed mouse cells or with T antigen positive somatic cell hybrids derived from SV40-transformed human and normal mouse parental cells. However, normal BALB/c mice readily produce antibodies to SV40 T antigen. When nude mice were reconstituted with normal syngeneic T lymphocytes from spleen or thymus source, the humoral immune responsiveness to SV40 T antigen was restored.     

Interleukin 2 induces interferon alpha/beta production in mouse bone marrow cells

We have previously reported that mouse bone marrow (BM) cells stimulated with alloantigen produce cytotoxic effector T-cell activity and produce interferon (IFN-)alpha/beta. In this report we show evidence suggesting that interleukin 2 (IL-2) may play a role in this IFN-alpha/beta production by alloantigen-stimulated BM cells. Alloantigen-induced IFN production by bone marrow cells was completely inhibited when cultures were supplemented with antisera to IL-2. Cell-free supernatants obtained at 2 days from cultures containing C57BL/6 BM cells and irradiated DBA/2J spleen cells were also shown to contain low levels of IL-2 activity and induced significant IFN production in fresh BM cells. Different IL-2 preparations were tested for their ability to induce IFN-alpha/beta production in mouse BM cells. Mouse BM cells cultured with recombinant human IL-2 or highly purified mouse IL-2 produced high levels of IFN-alpha/beta activity after 2-3 days of culture with significant IFN activity being detected as early as 24 hr of culture. IL-2-induced IFN-alpha/beta production was partially resistant to irradiation. In contrast, irradiated (2000 rad) bone marrow cells failed to produce any IFN when cultured with alloantigen in the absence of IL-2. T-cell-depleted BM cells or BM cells obtained from C57BL/10 nude mice produced high levels of IFN-alpha/beta following stimulation with IL-2. In addition, bone marrow cells depleted of Ia+, Qa 5+, or Asialo GM+1 cells produced IFN in response to IL-2. Thus, neither T cells nor NK cells are required for IL-2-induced IFN-alpha/beta production by BM cells. The action of IL-2 on bone marrow cells to induce IFN production was mediated by the classical IL-2 receptor, since monoclonal antibodies to the IL-2 receptor present on T cells blocked this response and since bone marrow cells depleted of IL-2 receptor-bearing cells failed to produce IFN when cultured with IL-2. These results suggest that non-T cells resident in the BM have receptors for IL-2 and can produce IFN-alpha/beta upon stimulation by IL-2. Since IFN has been shown to affect different aspects of hematopoiesis, the production of IFN by BM cells stimulated by IL-2 may be important in the control of hematopoiesis. In addition, IL-2-induced IFN production may play a role in graft-versus-host disease.     

Dysregulation of interleukin-10 and interleukin-12 are involved in the reduced host resistance to Listeria monocytogenes infection in alymphoplastic aly mutant mice

The aly is a unique spontaneous autosomal recessive mutation in mice that causes a systemic defect of lymph nodes and Peyer's patches and disorganized splenic and thymic structures with immunodeficiency. Our previous study demonstrated that resistance to Listeria monocytogenes infection and interferon-gamma (IFN-gamma) production are attenuated in the mutant mice. In this study, we investigated the mechanism of decrease in antilisterial resistance and IFN-gamma production in aly mice. Interleukin (IL)-12 production in response to heat-killed L. monocytogenes (HK-LM) was decreased but IL-10 production was increased in aly/aly macrophage cultures, compared with those in aly/+ macrophages. Nonadherent cells and macrophages obtained from the spleens of naive aly/+ mice and aly/aly mice were reconstituted and stimulated with HK-LM. IFN-gamma production was markedly decreased when macrophages derived from aly/aly mice were used. IFN-gamma production in aly/aly spleen cell cultures was recovered in the presence of anti-IL-10 monoclonal antibody (mAb) or recombinant IL-12. When aly/+ mice and aly/aly mice were injected with mAb against IL-10 or IL-12 p40, antilisterial resistance was inhibited by injection of anti-IL-12 p40 mAb, while anti-IL-10 mAb treatment augmented the resistance. Administration of anti-IFN-gamma mAb attenuated antilisterial resistance in aly/+ mice but not in aly/aly mice. The present results suggest that downregulation of IL-12 and upregulation of IL-10 in macrophages might be involved in the decrease in antilisterial resistance and IFN-gamma production in aly/aly mice in addition to the structural defect in lymphoid organs. Moreover, the results predict that an IL-12-dependent and IFN-gamma-independent mechanism may be also involved in the decrease in antilisterial resistance in aly/aly mice.     

Role of L3T4+ and LyT-2+ cells in experimental visceral leishmaniasis

In contrast to euthymic (nu/+) BALB/c mice, athymic nude (nu/nu) BALB/c mice fail to control the visceral intracellular replication of Leishmania donovani, do not generate the macrophage-activating lymphokine IFN-gamma, and show little or no granulomatous tissue response. To characterize the T cell requirement for successful defense against L. donovani, nude mice were first reconstituted with unfractionated nu/+ immune spleen cells, which readily conferred the capacity to control and eliminate visceral (hepatic) L. donovani. In reconstituted mice, acquired resistance was paralleled by the ability of spleen cells to generate high levels of leishmanial Ag-stimulated IFN-gamma and the development of well formed liver granulomas. In contrast, nude mice reconstituted with either L3T4+- or Lyt-2+-enriched immune spleen cells alone failed to control visceral parasite replication and did not develop effective granulomas despite the finding that transfer of L3T4+ cells largely and Lyt-2+ cells partially restored the capacity to secrete IFN-gamma. To determine whether both T cell subsets were also required in a normal host, nu/+ BALB/c mice were treated with cell-depleting anti-L3T4 and anti-Lyt-2 mAb. Depletion of either T cell subset inhibited the acquisition of resistance to L. donovani and impaired the tissue granulomatous response. Thus, successful T cell-dependent host defense towards intracellular L. donovani and the tissue expression (granulomas) of this mechanism appear to require both L3T4+ and Lyt-2+ cells. A primary role for the L3T4+ cell may be IFN-gamma production; the role of the Lyt-2+ cell and the precise interaction of the two T cell subsets remain to be identified.     

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.     

Induction of interferon production in mouse spleen cell cultures by corynebacterium parvum.

Spleen cells of CS7BL/6 mice produced considerable amounts of interferon (IF) in vitro when tested 5 to 20 days after injection of killed Corynebacterium parvum. Interferon was also produced when C. parvum was added in vitro to spleen cell cultures of previously untreated mice. High levels were detected after 1 day of culture with some increment during subsequent days. In a number of experiments IF was also produced in untreated control cultures but only after prolonged cultivation and not after 1 day. The highest levels of IF were usually obtained when spleen cells of C. parvum-treated mice were challenged with additional C. parvum in vitro. The IF induced by C. parvum shared certain physicochemical properties with a tested immune IF and was not neutralized by an antiserum raised against a type I IF. Spleen cells of nu/nu mice and spleen cells treated by anti-θ serum plus complement did not differ from their respective controls, indicating that production of IF did not require mature T lymphocytes. Removal of B lymphocytes by nylon wool columns abolished the capacity of spleen cells to produce IF. When spleen cells were freed of adherent cells by the use of plastic surfaces, they no longer produced IF. Peritoneal exudate macrophages (PEC), which by themselves did not produce IF, in small numbers reconstituted nonadherent spleen cells. Nylon column-treated spleen cells, however, could not be restored by PEC. It is concluded that IF upon challenge with C. parvum is produced by B lymphocytes and requires the help of macrophages.     

Simultaneous production of IFN-gamma, IFN-alpha/beta and nitric oxide in peritoneal macrophages from TDM-treated mice

Peritoneal macrophages (PM) were isolated from mice treated with Dimycolate of Trehalose (TDM), a glycolipid extracted from the cell wall of Mycobacterium tuberculosis. PM from TDM-treated mice (TDM-PM) were shown to secrete consistent amount of IFN-gamma, which was not detectable in control Resident-PM (Res-PM), as revealed by ELISA. In addition, biologically active IFN was detected in the supernatants of TDM-PM, whereas no IFN production was found in those of control Res-PM. The addition of specific antisera to PM cultures revealed the simultaneous production of both type I and II IFNs in TDM-PM cultures. No reciprocal regulation in the production of IFN-gamma and IFN-alpha/beta was found in these cultures. In parallel, nitric oxide (NO) production was measured in TDM-PM cultures by detecting nitrites (NO2-). TDM-PM cultures accumulated high amounts of NO2- which decreased to the level of Res-PM in the presence of NMMA, an inhibitor of NO-synthases. In vitro, neither type I nor type II IFNs were involved in the stimulation of NO production. The capacity of macrophages to simultaneously secrete IFN-gamma, IFN-alpha/beta and NO upon in vivo TDM-treatment could be of particular relevance for the defense process of innate immunity in which macrophages play a crucial role.     

Strain differences of interferon-generating capacity and resistance in toxoplasma-infected mice

To explore a possible correlation between susceptibility to Toxoplasma and interferon (IFN)-generating capacity in mice, we compared the levels of serum IFN induced by stimulation with Toxoplasma lysate antigen (TLA) in different strains of Toxoplasma-infected and uninfected mice. Injection of TLA into five strains of mice with chronic Toxoplasma infection resulted in the release of considerable amounts of IFN into the circulation. Most of these IFN activities were acid labile and not neutralized by sheep antiserum against mouse IFN-alpha/beta, indicating that IFN-gamma was the dominant form produced in this system. In contrast, the majority of IFN induced in uninfected mice was characterized as IFN-alpha/beta by their acid stability and antigenicity. The response of IFN production in Toxoplasma-infected and uninfected mice varied quantitatively depending on the mouse strains examined. C57BL/6 mice were found to be the best producers of both IFN-alpha/beta and IFN-gamma, while BALB/c mice were consistently poor producers of both IFN populations. A/J, DBA/2, and C3H/He mice could be roughly classified as intermediate producers of both IFN populations. C57BL/6 and C3H/He mice showed a significant prolongation of mean survival time following primary or secondary infection with Toxoplasma compared to that of BALB/c mice. However, there was no direct correlation between the susceptibility to Toxoplasma and the levels of serum IFN.     

Interferon-mediated protection of B16 melanoma cells from cytotoxicity by activated macrophages

Corynebacterium parvum-activated macrophages (M phi), purified by adherence, were cytotoxic for B16 melanoma cells maintained in vitro. Pretreatment of the melanoma cells for 18 hr with interferon-alpha/beta or -gamma (IFN-alpha/beta or -gamma) caused a reduced susceptibility of the B16 cells to M phi-mediated cytotoxicity. The IFN-induced protective effect of B16 cells from cytotoxic M phi was found to be dose dependent. In addition, IFN-gamma was more protective than IFN-alpha/beta. The protective effect observed with partially purified IFN was reproduced by using highly purified IFN-alpha/beta or recombinant IFN-gamma. Monoclonal antibodies to IFN-gamma neutralized the protective effect provided by IFN-gamma. These results show that the susceptibility of a tumor cell line to killing by activated M phi can be altered by IFN pretreatment.     

Interleukin-6 production in Mycobacterium bovis BCG-infected mice.

Mycobacterium bovis BCG and its subcellular components (bacterial extract, culture filtrate, purified protein derivative, and muramyl dipeptide MDP) are potent in vitro IL-6 inducers in spleen cell cultures from uninfected and BCG-infected BALB/c mice. Both plastic adherent and nonadherent spleen cells are capable of producing IL-6. Athymic nude mice produce more IL-6 than euthymic mice, suggesting that monocyte/macrophages are the main IL-6-producing cells in response to BCG. Finally, IL-6 production seems to be controlled to some extent by T lymphocytes, as down-regulation of CD4+ cells resulted in a marked increase in IL-6 production. Interferon-gamma does not seem to be involved in this regulation.