The role of tumor necrosis factor in viral disease.

Tumor Necrosis Factor (TNF) is one of the many cytokines that comprise a complex intertwined network of biological response modifiers that takes on extreme significance as the host response to infectious diseases. Soluble factors such as Interleukin-2 and Interferon-gamma released by T cells and Interleukin-1, Interleukin-6 and TNF released by monocytes have been shown to play key roles in proliferation, activation and differentiation of immune cells. It has also become evident that development of treatment modalities for infectious diseases is complicated by the complexity of this cytokine network. In the last decade numerous reports have presented data, often conflicting, which clearly demonstrate a role for TNF in the response to infections caused by viruses. This review summarizes this rapidly growing volume of data, discussing consistencies and discrepancies as appropriate. By better understanding the role of TNF in the host immune response, it may be possible to modulate this complex network for the benefit of the host in its battle against viral infection.

The effect of interferon-gamma on rejection and neutrophil function following transplantation.

Rats were used as a model for a living heterotopic cardiac allograft organ transplant. Rats treated in this model with recombinant rat interferon-gamma (IFN-gamma) showed accelerated rejection in a dose-dependent fashion. However, rats treated with maintenance doses of cyclosporine and IFN-gamma expressed increased rejection at 20 days that had resolved completely by 45 days post-transplantation. Polymorphonuclear leukocytes (neutrophils) were isolated from the blood of rats, and their function was determined by treating the cells with f-Met-Leu-Phe (fMLP) and measuring superoxide produced. Results indicate that the neutrophils from rats treated with maintenance doses of cyclosporine and IFN-gamma still had increased IFN-gamma-modulated fMLP-induced respiratory burst and that maintenance cyclosporine therapy can inhibit the IFN-gamma-mediated accelerated rejection without compromising the antimicrobial effects of IFN-gamma treatment.

Interferon-gamma and resistance to bacterial infections.

Since its initial description as an antiviral, it has become clear that Interferon-gamma (IFN-gamma) has potent immunoregulatory and cell growth regulatory activities. As a result of these additional activities, it is now apparent that IFN-gamma plays a major role in regulation of bacterial infections. IFN-gamma can be both induced by bacteria and bacterial products; endogenous IFN-gamma production has been shown to play a protective role in the natural host response to several bacterial infections; and administration of exogenous IFN-gamma is effective in the prevention and treatment of bacterial infections in numerous animal model systems. Although it is now clear that IFN-gamma plays a role in regulation of bacterial infections, the mechanisms of its anti-bacterial effects in vivo remain to be established due to the pleiotropic nature of IFN-gamma activity.

Effect of in vivo administration of recombinant murine gamma interferon on in vitro lymphoproliferative responses following immunization with Candida albicans.

The effect of in vivo administration of recombinant murine gamma interferon (rMuIFN-gamma) on in vitro proliferation of lymphocytes to Candida antigens and lectins was examined in naive CBA/J mice and in similar mice colonized with Candida albicans by intragastric (i.g.) intubation and/or inoculated intradermally (i.d.) with the fungus. Lymph node lymphocyte and splenic lymphocyte (splenocyte) responses to soluble cytoplasmic substances derived from C. albicans varied with the route of inoculation of the fungus, the sex of the animal, and the presence or absence of rMuIFN-gamma treatment. In the absence of rMuIFN-gamma treatment, lymphoid cells from lymph nodes draining the site of the i.d. lesion responded well to soluble cytoplasmic substances. Colonization of the gut of female mice with C. albicans either had no effect or promoted better lymph node responses when such animals were also challenged i.d., whereas gut colonization of males followed by i.d. challenge appeared to have a suppressive influence on the level of proliferation in response to antigens in vitro. Antigen-specific splenocyte responses could be detected as well, and they were best in animals inoculated i.g.-i.d. or i.d. only. With the exception of lymph node lymphocytes from male mice, treatment of infected animals, regardless of the route of infection, with rMuIFN-gamma frequently resulted in lowered responses to antigens when comparable treatment groups were examined. With respect to mitogen stimulation, infection with C. albicans, especially i.g. or i.g.-i.d., resulted in a population of lymph node lymphocytes with lower-than-normal responses to concanavalin A but higher-than-normal responses to lipopolysaccharide (LPS). Splenocyte responses to mitogens were not altered as dramatically as the responses of lymph node lymphocytes, but splenocytes from female mice had a suppressed response regardless of the route of exposure to C. albicans, and those from mice which were maximally stimulated with C. albicans, i.e., inoculated i.g.-i.d., also had a suppressed response to concanavalin A. Treatment with rMuIFN-gamma either had no effect on the subsequent splenocyte responses or boosted subnormal mitogen responses toward the normal range. Collectively, these data illustrate that exposure to both C. albicans and rMuIFN-gamma influenced the responses to mitogen and C. albicans antigen of lymph node lymphocyte and splenocyte populations, as detected in vitro by lymphoproliferation. Treatment with rMuIFN-gamma often resulted in increased responsiveness to a B cell mitogen, LPS, and decreased responsiveness to a C. albicans antigen.

Recombinant human interferon-gamma modulates Rift Valley fever virus infection in the rhesus monkey.

Prophylactic treatment of rhesus macaques with 10(4)-10(6) U/kg of recombinant human interferon-gamma (rHuIFN-gamma) modulated Rift Valley fever (RVF) virus infection. IFN was given intramuscularly at 24 h prior to infection and daily thereafter for a total of five doses. After infection, treated monkeys showed no evidence of clinical disease; some had no detectable viremia; when viremia was observed, peak virus titers were decreased compared to control infected monkeys; and only minor and transient perturbations in hematologic and clinical chemistry values were seen. Untreated infected control monkeys developed high-titered viremia, mild to severe clinical disease, and moderate to severe changes in hemostatic parameters and clinical laboratory measurements. No evidence of synergism was noted when RVF virus-infected monkeys were treated prophylactically with combined low doses of rHuIFN-gamma and rHuIFN-alpha A.

Interferon-gamma increases alveolar macrophage Ia antigen expression despite oral administration of dexamethasone to rats.

Corticosteroids have multiple effects on immune and inflammatory responses and decrease host resistance to a broad range of microorganisms. Resident tissue macrophages have been proposed as a target for the immunosuppressive effects of corticosteroids and are important in host defense against infections. During infection-induced immune responses, macrophages are activated after exposure to interferon-gamma (IFN-gamma), and class II major histocompatibility (Ia) antigens on their surface are increased. We investigated the effect of orally administered corticosteroids on alveolar macrophages, the resident macrophages of the lung parenchyma. We hypothesized that corticosteroids would inhibit the activation of alveolar macrophages and measured induction by IFN-gamma of Ia antigens as a marker of cell activation. Alveolar macrophages from normal and corticosteroid-treated rats were exposed to recombinant murine IFN-gamma (rMuIFN-gamma) in vitro and assayed for Ia transcription and surface Ia expression. Ia mRNA accumulation was induced in alveolar macrophages from normal and corticosteroid-treated rats after exposure in vitro to rMuIFN-gamma. Furthermore, rMuIFN-gamma increased surface expression of Ia proteins on alveolar macrophages from corticosteroid-treated rats, although to a lesser extent than on cells from control rats. Finally, surface Ia expression could also be increased in vivo by exposure of corticosteroid-treated rats to an aerosol containing rMuIFN-gamma. These results demonstrate that administration of oral corticosteroids, while establishing a state of immunosuppression in rats, does not abolish responsiveness of rat alveolar macrophages to rMuIFN-gamma. We speculate that IFN-gamma-induced augmentation of phagocytic cell function may constitute an important therapeutic modality to treat complications of immunodeficiency.

In vivo immune responses to Candida albicans modified by treatment with recombinant murine gamma interferon.

The immunologic effects of in vivo administration of recombinant murine gamma interferon (rMuIFN-gamma) were determined in a murine model of candidiasis. Naive mice were given graded doses of rMuIFN-gamma and then challenged intravenously with Candida albicans. Increased morbidity and mortality were noted in four different strains of mice, viz., BALB/c, A/J, Swiss Webster, and CBA/J, providing the mice had not been immunized with C. albicans before challenge. Quantitative culture of selected organs of Swiss Webster and CBA/J mice surviving treatment with rMuIFN-gamma revealed elevated numbers of C. albicans cells, particularly in the kidneys, but also in the liver, lungs, and spleen. The lungs, livers, and spleen of female CBA/J mice were more protected from increased multiplication of the fungus than were those of males of the same species or female Swiss Webster mice. On the basis of these initial findings, the effect of treatment with 5,000 U of rMuIFN-gamma on immune responses in a gastrointestinal model of candidiasis was determined. CBA/J mice that had been colonized with C. albicans as infants were boosted with a cutaneous inoculation of the fungus when 6 to 10 weeks old; development of delayed hypersensitivity (DH), antibodies, and protective responses was assayed at intervals thereafter. Daily treatment with rMuIFN-gamma (beginning 1 day before cutaneous inoculation) suppressed weak immune responses but had little effect on responses which were strong. For example, DH and anti-C. albicans antibody production were suppressed in animals colonized with C. albicans but not boosted by cutaneous inoculation, and DH was suppressed in uncolonized animals that had been inoculated once cutaneously with the fungus as well. There was no rMuIFN-gamma-induced suppressive effect of DH in mice which had been stimulated maximally with C. albicans, i.e., colonized animals that had been boosted cutaneously with the organisms. Collectively, these data indicate that naive mice or mice with minimal levels of anti-C. albicans sensitivity, females somewhat more so than males, were sensitive to suppressive effects of in vivo treatment with rMuIFN-gamma when challenged with C. albicans. In contrast, under conditions similar to those of humans, in whom underlying immunity to C. albicans is usually present, suppression of host responses to C. albicans was not observed in immunized mice in response to treatment with rMuIFN-gamma.

In vitro treatment of HEp-2 cells with human tumor necrosis factor-alpha and human interferons reduces invasiveness of Salmonella typhimurium.

Enteroinvasive bacteria, like Salmonella typhimurium, can be internalized in in vitro cultured epithelioidal cells, like HEp-2 cells. This phenomenon is inhibited by pretreatment of cells with human tumor necrosis factor alpha (TNF-alpha) in a dose- and time-dependent manner. The effect was also reproduced in other cell types, including diploid embryo fibroblast cells. The TNF-alpha effect was neutralized by anti-TNF-alpha antibodies. No synergistic effect was produced by combinations of TNF-alpha with either interferon alpha or gamma. Unlike the effects of interferons, TNF-alpha inhibited the invasiveness of Shigella flexneri and the TNF-alpha effect was not inhibited by cycloheximide.

Role of endogenous gamma interferon in host defense against Chlamydia trachomatis infections.

BALB/c mice (6 to 8 weeks old) infected with Chlamydia trachomatis serovar L1 were sacrificed, and the yield of Chlamydia inclusion-forming units from the liver and lungs was measured in HeLa 229 cells. The yield of inclusion-forming units reached a peak at 3 days postinfection and then progressively declined. The mice infected with C. trachomatis had no detectable levels of gamma interferon (IFN-gamma) in their sera. However, stimulation of their spleen cells with either concanavalin A or heat-killed C. trachomatis resulted in the release of high levels of IFN-gamma (600 to 900 IU/ml) at 5 to 8 days postinfection. The increased release of IFN-gamma from the spleen cells paralleled the clearance of chlamydia from the liver and lungs. Sera and spleen cells from animals immunized with live C. trachomatis were transferred to recipient mice that were subsequently challenged with C. trachomatis. Transfer of spleen cells resulted in a reduction of the infection in the recipient animal as measured by the yield of chlamydia from the spleen, but transfer of the sera did not confer protective immunity. In addition, mice infected with C. trachomatis serovar L1 were treated with a hamster neutralizing monoclonal antibody to recombinant murine IFN-gamma (MAb-MuIFN-gamma). In the animals receiving the MAb-MuIFN-gamma, the yield of chlamydia from the lungs, spleen, and liver was significantly higher than from the control groups of mice. Histopathological analysis of tissues from the chlamydia-infected mice showed that the animals treated with the MAb-MuIFN-gamma had a significantly more extensive inflammatory reaction in their lungs, liver, and spleen.

Transforming growth factor-beta 1 modulates the expression of class II histocompatibility antigens on human cells.

We have examined the effects of transforming growth factor-beta (TGF-beta 1) on the expression of the class II histocompatibility Ag, HLA-DR: 1) induced by human rIFN-gamma (rHuIFN-gamma) in human melanoma, Hs294T cells and 2) constitutively expressed in a subclone Hs294T cell line. The expression of HLA-DR Ag on Hs294T cells was induced by rHuIFN-gamma in a dose- and time-dependent manner with maximal levels obtained with 10 ng/ml rHuIFN-gamma after 48 h exposure. Treatment of Hs294T cells with natural porcine platelet-derived or human rTGF-beta 1 (1 to 100 ng/ml) in the presence of rHuIFN-gamma (0.1 to 10 ng/ml) reduced the percentage of cells positive for this Ag by approximately 40 to 50%, as determined by flow cytometry. This suppressive effect of TGF-beta 1 was not restricted to Hs294T cells inasmuch as rHuIFN-gamma induced HLA-DR surface Ag expression on PBMC-derived adherent cells was also significantly inhibited by TGF-beta 1. Northern blot analysis of cytoplasmic RNA extracted from Hs294T cells indicated that the decreased levels of HLA-DR cell-surface Ag correlated with decreased levels of mRNA transcripts encoding this Ag. TGF-beta 1 also effectively reduced the levels of HLA-DR mRNA in 1) cells that had been pretreated with rHuIFN-gamma and were expressing maximal levels of HLA-DR surface antigen and 2) cells that constitutively expressed HLA-DR surface Ag without exogenous rHuIFN-gamma treatment.

Recombinant murine gamma interferon inhibits Chlamydia trachomatis serovar L1 in vivo.

Chlamydia trachomatis serovar L1 injected intravenously in mice resulted in systemic nonlethal infections of the animals. Treatment of mice with recombinant murine gamma interferon resulted in a decrease in the number of infectious C. trachomatis organisms recovered from the lungs, spleens, and livers as well as in a decrease of the inflammatory reaction in those organs when assessed 3 and 5 days after challenge.

The antichlamydial, antiviral, and antiproliferative activities of human gamma interferon are dependent on the integrity of the C terminus of the interferon molecule.

The effects of recombinant human gamma interferon (rHuIFN-gamma; two identical monomers of 140 residues in length) and of two re-engineered C-terminal variants, rHuIFN-gamma Tetra-Ser (residues 129 to 132 replaced by serine) and rHuIFN-gamma 125 (two identical monomers of 125 residues each with the last 14 residues plus an additional alanine from the C terminus deleted), were compared in terms of several in vitro biological activities. By using three different human cell lines (HeLa 229, HEp-2, and A549), the interferons were tested for their ability to inhibit: (i) growth of Chlamydia trachomatis; (ii) replication of encephalomyocarditis virus; and (iii) cell growth. rHuIFN-gamma restricted the growth of chlamydiae to 50% of the non-IFN-treated control at concentrations ranging from 0.01 to 0.05 ng/ml, depending on the cell type assayed. One of the modified proteins, rHuIFN-gamma Tetra-Ser, also decreased the growth of chlamydiae, but it required a concentration of approximately 0.5 ng/ml to produce 50% inhibition. rHuIFN-gamma 125 had the lowest antichlamydial activity of the three IFN-gamma variants tested; concentrations of 1 to 20 ng/ml were needed to reduce the growth of C. trachomatis to 50% of that of the control. The relative antiviral and antiproliferative activities of the three IFN-gamma preparations paralleled their antichlamydial activities in these three cell lines. The antiencephalomyocarditis virus activities of rHuIFN-gamma Tetra-Ser and rHuIFN-gamma 125 were reduced by approximately 10-fold and 10(2)- to 10(3)-fold, respectively, compared with the antiviral activity of rHuIFN-gamma. Proliferation of the three cell lines was restricted to approximately 50% of the control with 0.5 to 10 ng of rHuIFN-gamma per ml. Inhibition of cell growth by rHuIFN-gamma Tetra-Ser was significant only at concentrations equal to or greater than 30 ng/ml, and the rHuIFN-gamma 125 variant did not significantly decrease the growth of any of the three cell lines at the concentrations tested. These results suggest that the C-terminal portion of rHuIFN-gamma is critical for maintaining the conformation necessary for inducing the antichlamydial, antiviral, and antiproliferative activities of the molecule.

Ultrastructural analysis of the anti-chlamydial activity of recombinant murine interferon-gamma.

The effect of murine interferon-gamma (MuIFN-gamma) on the developmental cycle of Chlamydia trachomatis in McCoy cells was analyzed by light and electron microscopy. Addition to the culture media of 10 ng/ml of MuIFN-gamma, either 24 hr before or immediately after Chlamydia infection, resulted in a significant inhibition of the growth of this organism. Microscopic analysis showed that with both treatments the majority of microorganisms were arrested at the elementary body stage. Only a few small chlamydial inclusions were detected at 48 hr postinfection and contained predominately reticulate bodies. Furthermore, the growth of Chlamydia was arrested in cells that were treated with MuIFN-gamma at various intervals following infection. Addition of MuIFN-gamma at 8 or 12 hr after infection resulted in the arrest of chlamydial growth before initiation of reticulate body fission. When the MuIFN-gamma was added 24 hr postinfection, we could detect, by electron microscopy, inhibition at the stage of reticulate body replication.

Recombinant human gamma interferon inhibits simian malaria.

Prophylactic treatment with 0.1 mg of human gamma interferon per kg (body weight) per day completely suppressed experimental infection with Plasmodium cynomolgi B sporozoites in rhesus monkeys. Treatment with lower doses partially suppressed this infection. Prophylactic treatment with human gamma interferon, however, had no protective effect against trophozoite-induced infection, suggesting that the interferon effect was limited to the exoerythrocytic stage of parasitic development.

Levels of interferon in blood serum and toxicity studies of bacteria-derived bovine alpha I1 interferon in dairy calves.

This paper reports information on the levels of interferon (IFN) in the blood serum of dairy calves given 10(6) U of bacteria-derived bovine alpha I1 interferon per kg of body weight by intravenous (i.v.), intramuscular (i.m.), subcutaneous (s.c.), and intranasal (i.n.) routes. Highest levels (10,000 U/ml) in the vesicular stomatitis viral assay system were obtained after i.v. administration and occurred within 30 min of a dose; levels rapidly declined thereafter to a low of 200 to 300 U/ml by 24 h. Serum inhibitory activity against vesicular stomatitis virus in this range is sometimes found in normal dairy calves. Levels after i.m. and s.c. administration were similar: a plateau of 1,000 to 2,000 U/ml between 2 and 8 h after a treatment with a decline to 200 to 300 U/ml by 24 h. Serum IFN was not detected after i.n. dosing or in the control group given physiological buffered saline by the i.m. route. A transitory moderate febrile response, but no other clinical adverse effects, was noted after the first intramuscular dose of IFN, but not after subsequent i.m. doses. No clinical signs were noted after i.v., s.c., or i.n. dosing or in the control calves given physiological buffered saline intramuscularly. After i.v., s.c., and i.m. administration of IFN, leukopenia, neutropenia, and lymphocytopenia were observed; these were most prominent within the first 24 h after the initial dose of IFN.

In vitro biological activities of Escherichia coli-derived bovine interferons-alpha, -beta, and -gamma.

The infectious yields of several bovine viruses were inhibited in bovine cells treated with purified preparations of E. coli-derived bovine interferons (BoIFNs)-alpha, -beta, and -gamma. BoIFN-beta 2, encoded by one member of the BoIFN-multigene family, had more potent antiviral and antiproliferative activities than the product of one member of the class-I IFN-alpha gene family (BoIFN-alpha I1). BoIFN-beta 2 also completed more effectively than BoIFN-alpha I1 with HuIFN-alpha I2 for cell-surface receptors on bovine cells. Despite these differences, the kinetics of maximal antiviral activity were similar for BoIFN-alpha I1, BoIFN-beta 2, and HuIFN-alpha I2. In comparison to BoIFN-alpha I1 and BoIFN-beta 2, BoIFN-gamma had the least in vitro antiviral activity and required the longest contact with cells to achieve maximal protection against virus infection, but had a dramatically greater antiproliferative activity.

The anti-chlamydial and anti-proliferative activities of recombinant murine interferon-gamma are not dependent on tryptophan concentrations.

The effect of recombinant murine interferon-gamma on the growth of Chlamydia trachomatis was analyzed in a mouse fibroblast cell line (McCoy cells). Murine interferon-gamma had a very potent anti-chlamydial activity, although minimally affecting cellular proliferation. Over 95% inhibition of chlamydial inclusions was obtained at a concentration of 1 U/ml of interferon. At a concentration of 1 U/ml of murine interferon-gamma, there was minimal inhibition of the proliferative capacity of McCoy cells. Approximately 50% inhibition of cell growth was obtained with a concentration of 10 U/ml of interferon. Varying concentrations of tryptophan in the medium did not alter either the anti-chlamydial or the anti-proliferative activity of the interferon.

Effect of bovine alpha 1 interferon on bovine herpesvirus type 1-induced respiratory disease.

Treatment of calves with bovine recombinant alpha 1 interferon prior to challenge with bovine herpesvirus type 1 increased the animals' ability to withstand a subsequent Pasteurella haemolytica challenge. The reduction in viral-bacterial synergy observed following interferon treatment did not appear to be due to a direct effect of the interferon on virus replication in the upper respiratory tract. Thus, even though interferon-treated animals shed slightly less virus from their nasal passages than did untreated animals, this reduction was not statistically significant. Furthermore, there was no difference in the level of intranasal interferon secreted by control or interferon-treated animals. These results suggest that interferon treatment does not affect the production of endogenous interferon. In contrast, a significant difference was observed between the number of days that control animals were sick, the levels of serum fibrinogen and the functional activity of polymorphonuclear neutrophilic granulocytes obtained from infected calves. These results suggest that bovine recombinant alpha 1 interferon may have a greater immunomodulatory effect than a direct antiviral effect in this model. This is further supported by the observation that bovine herpesvirus type 1 is relatively resistant to the direct antiviral effect of bovine recombinant alpha 1 interferon in vitro.