Role of interferon alpha (IFN-alpha) and interferon gamma (IFN-gamma) in the control of the infection of monocyte-like cells with human cytomegalovirus (HCMV)

The role of cytokines in the control of HCMV infection has been studied in THP-1 cells, a macrophage-like cell model and in MRC-5 cells. HCMV replication was studied by immune detection of viral immediate-early antigens (IEA) and virus yield was evaluated in MRC-5 cells by immunoperoxidase staining. Pretreatment of MRC-5 and phorbol 12-myristate 13-acetate (PMA)-treated THP-1 cells with IFN-alpha or IFN-gamma for 24 hr prior to the infection reduced the number of infected cells and virus yield. A synergistic anti-CMV activity in synthesis of early proteins was obtained with these cytokines in combination with TNF-alpha in differentiated THP-1 cells only. Treatment of HCMV-infected differentiated THP-1 cells or MRC-5 cells with IFN-alpha or IFN-gamma alone had no inhibitory effect on virus replication, however the virus yield was reduced with ganciclovir. A synergistic anti-CMV activity in virus yield was obtained only when infected differentiated THP-1 cells were treated with ganciclovir in combination with IFN-gamma. The current study shows that IFN-alpha and IFN-gamma can play a role in the reduction of HCMV replication in macrophage-like cells and in the efficiency of therapies with ganciclovir in this cell type and that the anti-CMV effect of cytokines may be different in fibroblasts and in macrophage-like cells.     

Immunologic monitoring studies in advanced cancer patients treated with recombinant human gamma interferon (IFN-gamma 4A)

Thirty-nine patients with a variety of advanced malignancies were treated with recombinant IFN-gamma 4A (AMGen, specific activity 1 to 5 x 10(7) U/mg protein). IFN-gamma 4A was administered at a dose of 10-2,000 micrograms/m2/d. Following a 2-week rest, a maintenance phase was continued with injections 3 d/wk. Immunologic monitoring studies were performed on patients' peripheral blood cells before administration of IFN-gamma 4A, then on Days 15 and 90. Flow cytometric analysis was used to determine the absolute number of CD 3+, CD 4+, CD 8+, CD 19+, and CD 16+ cells using a panel of monoclonal antibodies. Natural killer (NK) cell function was assayed by monitoring lysis of the K562 cell line in the Cr51 release assay. Changes from baseline were observed on Days 15 and 90 in all parameters studied, although the ratio of helper to suppressor cells seemed to remain within the normal range. Whereas there were no substantial changes in CD 3+ and CD 4+ cells on Day 15, IFN-gamma 4A had an enhancing effect on CD 8+, CD 19+, and CD 16+ cells. This trend continued at Day 90 only for CD 19+ and CD 16+ cells at the higher dose levels. An increase in functional NK cell activity at Day 15 was less noted on Day 90. Comparison of intravenous (IV) to intramuscular-subcutaneous (IM-SC) administration showed differences in the effect on lymphocyte subpopulations at 450 and 1,000 micrograms. The effect of IFN-gamma 4A on the equilibrium among lymphocyte subpopulations and the possibility of its role in combination therapy with other biologic response modifiers are discussed.     

Gamma interferon (IFN-gamma) receptor null-mutant mice are more susceptible to herpes simplex virus type 1 infection than IFN-gamma ligand null-mutant mice

Mouse strains with null mutations in the gamma interferon gene (Ifng) or the gamma interferon receptor gene (Ifngr) have been engineered. The use of these strains as animal models of viral and bacterial infections has enhanced our understanding of the role of gamma interferon (IFN-gamma) in the host immune response. However, direct comparisons between Ifng-/- (GKO) and Ifngr-/- (RGKO) mice have been problematic because previously available strains of these mice have had different genetic backgrounds (i.e., C57BL/6 and BALB/c for GKO mice and 129/Sv//Ev for RGKO mice). To enable direct comparison of herpes simplex virus type 1 (HSV-1) infections in GKO and RGKO mice, we introduced the IFN-gamma null mutation into the 129/Sv//Ev background. We report that, after HSV-1 inoculation, mortality was significantly greater in RGKO mice than in GKO mice (38 versus 23%, P = 0.0001). Similarly, the mortality from vaccinia virus challenge was significantly greater in RGKO mice than in GKO mice. With differences in genetic background excluded as a confounding issue, these results are consistent with the existence of an alternative ligand(s) for the IFN-gamma receptor that is also capable of mediating protection against viral challenge.     

In vitro analysis of interferon gamma (IFN-gamma) and interleukin-12 (IL-12) production and their effects in ileal Crohn's disease

Crohn's disease is an inflammatory disease of the gut in which tumour necrosis factor (TNF) and T helper 1 (Th1) cytokines (interleukin (IL)-12, interferon (IFN)-gamma) are thought to play a major role. After the successes obtained with neutralisation of TNF, interest is now growing for therapy aiming at neutralisation of Th1-associated cytokines. Since cytokines are linked in a delicate network, in vitro cultures of ileal lamina propria mononuclear cells (LPMC) were set up for evaluation of a) IFN-gamma and IL-12 production, b) effects of rhIFN-gamma and rhIL-12 and c) effects of anti-IFN-gamma and anti-IL-12 on pro-inflammatory cytokines and IL-10 production. LPMC were isolated from surgical specimens of a total of 27 Crohn's disease and 17 caecum carcinoma (control) patients. Cells were stimulated with CD40L (which triggers myeloid CD40-expressing cells) or anti-CD3 +CD80 (which triggers T cells). LPMC from involved ileal, Crohn's disease produced, in both non-stimulated and stimulated conditions, more IFN-gamma and IL-12p70 than LPMC from non-involved tissue or from control patients. rhIFN-gamma significantly enhanced TNF production in both controls and in ileal Crohn's disease patients, while rhIL-12 enhanced IFN-gamma but not TNF production. LPMC from involved tissue were more sensitive to IL-12 than control LPMC. LP-T cell-dependent activation of monocytes was then studied by co-culture of anti-CD3/CD80-stimulated LPMC with fresh monocytes, which resulted in high IL-12, IFN-gamma, TNF and IL-10 production. The data show that neutralisation of either IL-12 or IFN-gamma with mAb in these cultures also affects secretion of the reciprocal cytokine and (in the case of anti-IL-12) also that of the anti-inflammatory cytokine IL-10. However, no effect of anti-IL-12 or anti-IFN-gamma on production of TNF, a cytokine with an important pathogenic role in Crohn's disease, could be found. Therapies aiming at neutralisation of IFN-gamma or IL-12 are therefore unlikely to replace anti-TNF, but they might provide an additive or synergistic effect.     

Primary hepatocytes from mice treated with IL-2/IL-12 produce T cell chemoattractant activity that is dependent on monokine induced by IFN-gamma (Mig) and chemokine responsive to gamma-2 (Crg-2)

The IFN-gamma-inducible proteins monokine induced by IFN-gamma (Mig) and chemokine responsive to gamma-2 (Crg-2) can contribute to IL-12-induced antiangiogenic and leukocyte-recruiting activities, but the extent to which leukocytes vs parenchymal cells in different organs contribute to the production of these molecules remains unclear. The results presented herein show that IFN-gamma-dependent induction of Mig and Crg-2 gene expression can occur in many nonlymphoid organs, and these genes are rapidly induced in purified hepatocytes isolated from mice treated with IL-2 plus IL-12, or from Hepa 1-6 hepatoma cells treated in vitro with IFN-gamma. In addition to depending on IFN-gamma, the ability of IL-12 or IL-2/IL-12 to induce Mig and Crg-2 gene expression in purified hepatocytes also is accompanied by the coordinate up-regulation of the IFN-gamma R alpha and beta-chains, in the absence of IL-12R components. Supernatants of primary hepatocytes obtained from mice treated in vivo with IL-2/IL-12 or from hepatocytes treated in vitro with IFN-gamma contain increased chemotactic activity for enriched human and mouse CD3(+) T cells, as well as mouse DX5(+) NK cells. The hepatocyte-derived chemotactic activity for mouse T cells but not NK cells was ablated by Abs specific for Mig and Crg-2. These results suggest that parenchymal cells in some organs may contribute substantially to initiation and/or amplification of inflammatory or antitumor responses.     

Expression and characterization of recombinant interferon gamma (IFN-gamma) from the nine-banded armadillo (Dasypus novemcinctus) and its effect on Mycobacterium leprae-infected macrophages

Armadillos (Dasypus novemcinctus) manifest the full histopathological spectrum of leprosy, and are hosts of choice for in vivo propagation of Mycobacterium leprae. Though potentially useful as a model of leprosy pathogenesis, few armadillo-specific reagents exist. We have identified a region of high homology to the interferon gamma (IFN-γ) of other mammals within the recently published armadillo whole genomic sequence. cDNA was made from ConA-stimulated armadillo peripheral blood mononuclear cells (PBMC), amplified, and cloned into a pET expression vector for transformation and over-expression in Escherichia coli. The recombinant protein (rDnIFN-γ) was characterized by western blot and its biological function confirmed with bioassays including intracellular killing of Toxoplasma gondii and induction of indoleamine 2, 3-dioxygenase activity. In using rIFN-γ to activate macrophages from mice, humans or armadillos, similar to humans, rIFN-γ-activated armadillo MΦ did not produce nitrite and or inhibit the viability of M. leprae in vitro. Conversely, murine rIFN-γ-activated mouse MΦ produced high levels of nitrite and killed intracellular M. leprae in vitro. These data indicate that the response of armadillo MΦ to rDnIFN-γ is similar to that which occurs in humans, and demonstrates a potentially important value of the armadillo as a model in leprosy research.     

Essential pathogenetic role for interferon (IFN-)gamma in concanavalin A-induced T cell-dependent hepatitis: exacerbation by exogenous IFN-gamma and prevention by IFN-gamma receptor-immunoglobulin fusion protein

We have studied the effects of either exogenously-administered interferon (IFN-)gamma or of a nonimmunogenic mouse IFN-gamma receptor-Immunoglobulin (IFN-gamma R-Ig) fusion protein on the development of Concanavalin (Con)A-induced hepatitis in NMRI mice. PBS-treated control mice injected with 20 mg/kg ConA developed classical serological and histological signs of hepatitis with elevation of transaminases in the blood and infiltration of the liver by mononuclear cells and neutrophils. Treating the mice with rat IFN-gamma 24 h prior to and 1 h after ConA-challenge markedly exacerbated these signs of hepatitis in a dose-dependent fashion. Moreover, mice injected with lower, non hepatitogenic, doses of ConA (10, 5 mg/kg) became fully susceptible to develop hepatitis upon similar treatment with IFN-gamma. Concordantly, ConA-induced hepatitis was abrogated by either IFN-gamma R-Ig fusion protein or anti-IFN-gamma mAb. These data provide further evidence for the central pathogenetic role of endogenous IFN-gamma in ConA-induced hepatitis and demonstrate the feasibility to prevent disease development by means of a non immunogenic IFN-gamma R-Ig fusion protein.     

Exogenous interleukin-12 (IL-12) exacerbates Cryptosporidium parvum infection in gamma interferon knockout mice

Experimental infection of BALB/c- or C57BL/6-gamma-interferon-knockout (GKO) mice with Cryptosporidium parvum results in infection in both strains with different outcomes of disease. The BALB/c-GKO mice recover from infection, whereas the C57BL/6-GKO mice succumb to infection in less than 2 weeks. Differences in cytokine mRNA expression suggested that recovery may involve other cytokines. To determine whether the addition of either a Th1 or Th2 cytokine could alter the outcome of infection, we treated GKO mice with either recombinant (r)IL-4 or rIL-12 1 day before infection (DBI) or daily. No effect on the oocyst shedding patterns in either strain nor an increase in survival of the C57BL/6-GKO mice was observed in the rIL-4-treated mice. Whereas one dose of 0.5 microg rIL-12 given 1 DBI had no effect on oocyst shedding, we found that daily doses of rIL-12 administered intraperitoneally exacerbated C. parvum infection in both animal models. Administration of rIL-12 shortened the survival time in the C57BL/6-GKO mice and prevented BALB/c-GKO mice from recovering from infection. Specific proliferation of T cells to cryptosporidial antigen and Th1 and Th2 mRNA cytokine expression was markedly decreased in rIL-12-treated mice. Nitric oxide (NO) may have played a minor role in the decreased proliferation observed since levels of NO present in the splenocyte cultures from rIL-12-treated mice in response to parasite antigen stimulation were higher than those observed in controls. Thus, we propose that resistance to and recovery from C. parvum infections involves a fine balance in the amount and timing of Th1 and Th2 cytokines.     

Temporal expression of fumonisin B(1)-induced tumor necrosis factor-alpha and interferon gamma in mice

Fumonisin B(1) (FB(1)), a toxic metabolite of Fusarium verticillioides, is a carcinogen and causative agent of various animal diseases. Our previous studies indicated the involvement of tumor necrosis factor-alpha (TNF alpha) in FB(1)-induced toxic responses. To further investigate the time-course of TNF alpha production and signaling, mice (four/group) were treated subcutaneously (s.c.) or per os (p.o.) with either vehicle or 25 mg/kg of FB(1) as a single dose and sacrificed at 0, 2, 4, 8, 12 and 24 h after treatment. The TNF alpha expression was increased in liver and kidney after both routes of FB(1) exposure without any alterations in spleen. The p.o.-route FB(1) treatment caused greater hepatotoxicity compared to the s.c. route, as depicted by increased alanine aminotransferase and aspartate aminotransferase level in plasma, observed only after p.o. FB(1) treatment. The increase in enzymes at 8 h after p.o. treatment correlated with the highest TNF alpha expression, also noted at 8 h after p.o. treatment, thus further confirming the involvement of TNF alpha in FB(1) toxicity. The interferon (IFN)-gamma expression was increased in liver at 4 h after p.o. FB(1) treatment, suggesting a possible combined role of TNF alpha and IFN gamma in their induction and hepatotoxicity.     

High-yield amplification of Cryptosporidium parvum in interferon gamma receptor knockout mice

To date, large-scale production of Cryptosporidium parvum oocysts has only been achieved by amplification in neonatal calves and sheep. Many laboratories currently depend on supplies from external sources and store oocysts for prolonged periods which results in progressive loss of viability. Six to 8-week-old interferon gamma receptor knockout (IFN gamma R-KO) mice on a C57BL/6 background were inoculated by gavage (2000 oocysts/animal). Fecal pellets were collected daily from 7 days post-infection (p.i.) up to 2 weeks p.i. Intestinal oocyst yield was assessed at days 11, 12 and 14 p.i. by homogenization of intestinal tissues. Ether extraction and one or more NaCl flotations were used to purify oocysts. Total recoveries averaged 2.6 x 10(6) oocysts/mouse from fecal material and 3.8 x 10(7) oocysts/mouse from intestinal tissues. Overall, 2.3 x 10(9) purified oocysts were obtained from 60 mice. Recovered oocysts were capable of sporulation and were shown to be infectious both in vitro and in vivo. Oocyst amplification was achieved in only 11-14 days with minimal expense. The simplicity of this method presents a practical alternative for the routine passage, maintenance and storage of C. parvum in biomedical laboratories.     

IL-12 administration accelerates autoimmune diabetes in both wild-type and IFN-gamma-deficient nonobese diabetic mice,revealing pathogenic and protective effects of IL-12-induced IFN-gamma

IL-12 administration to nonobese diabetic (NOD) mice induces IFN-gamma-secreting type 1 T cells and high circulating IFN-gamma levels and accelerates insulin-dependent diabetes mellitus (IDDM). Here we show that IL-12-induced IFN-gamma production is dispensable for diabetes acceleration, because exogenous IL-12 could enhance IDDM development in IFN-gamma-deficient as well as in IFN-gamma-sufficient NOD mice. Both in IFN-gamma(+/-) and IFN-gamma(-/-) NOD mice, IL-12 administration generates a massive and destructive insulitis characterized by T cells, macrophages, and CD11c(+) dendritic cells, and increases the number of pancreatic CD4(+) cells secreting IL-2 and TNF-alpha. Surprisingly, IL-12-induced IFN-gamma hinders pancreatic B cell infiltration and inhibits the capacity of APCs to activate T cells. Although pancreatic CD4(+) T cells from IL-12-treated IFN-gamma(-/-) mice fail to up-regulate the P-selectin ligand, suggesting that their entry into the pancreas may be impaired, T cell expansion is favored in these mice compared with IL-12-treated IFN-gamma(+/-) mice because IL-12 administration in the absence of IFN-gamma leads to enhanced cell proliferation and reduced T cell apoptosis. NO, an effector molecule in beta cell destruction, is produced ex vivo in high quantity by pancreas-infiltrating cells through a mechanism involving IL-12-induced IFN-gamma. Conversely, in IL-12-treated IFN-gamma-deficient mice, other pathways of beta cell death appear to be increased, as indicated by the up-regulated expression of Fas ligand on Th1 cells in the absence of IFN-gamma. These data demonstrate that IFN-gamma has a dual role, pathogenic and protective, in IDDM development, and its deletion allows IL-12 to establish alternative pathways leading to diabetes acceleration.     

IFN-gamma induces expression of Fc gamma RIII (CD16) on human eosinophils.

The extent to which eosinophils constitutively express FcRIII (CD16) is controversial. We were unable to detect this receptor on freshly isolated, peripheral blood eosinophils. The capacity of eosinophils to change their Fc gamma R expression in vitro has not been previously demonstrated. Culture with IFN-gamma for 1 to 2 days induced FcRIII expression on eosinophils. This effect was dose-dependent and significant at concentrations of 100 U/ml IFN-gamma and above. Expression of FcRI (CD64) and FcRII (CDw32) was also upregulated. These increases were inhibited by cycloheximide (10(-6) M), suggesting a requirement for protein synthesis, and dexamethasone (10(-6) M). Northern blot analysis demonstrated the presence of FcRIII mRNA in eosinophils cultured with IFN-gamma for 2 days but not in unstimulated eosinophils. By contrast, culture with IL-3 caused an up-regulation of eosinophil FcRII expression but did not induce expression of FcRI or FcRIII. The FcRIII expressed by eosinophils after IFN-gamma stimulation was functionally active, as shown by the triggering of eosinophil membrane depolarization and LTC4 generation by an anti-CD16 mAb. Treatment of IFN-gamma-stimulated eosinophils with phosphatidylinositol-specific phospholipase C reduced FcRIII expression, suggesting that, like neutrophils, eosinophils express the phosphatidylinositol glycan-linked form of this receptor. Therefore, this study demonstrates that IFN-gamma-treated eosinophils express a functionally active, phosphatidylinositol glycan-anchored form of FcRIII.     

Temporal effects of gamma interferon deficiency on the course of Friend retrovirus infection in mice

The current studies demonstrate complex and seemingly contradictory effects by gamma interferon (IFN-gamma) on Friend virus (FV) infection. Both temporal and tissue-specific effects were observed. During the first week of infection, IFN-gamma-deficiency caused increased levels of FV infection in multiple tissues. Surprisingly, however, by 2 weeks postinfection, IFN-gamma-deficient mice had significantly lower levels of infection in both the spleen and bone marrow compared to wild-type mice. The rapid reduction of virus in the IFN-gamma-deficient mice correlated with a more rapid virus-neutralizing antibody response than was observed in the wild-type mice. Furthermore, the virus-neutralizing antibody response in wild-type mice could be accelerated by ablation of their IFN-gamma response. Although the IFN-gamma-deficient mice developed an accelerated virus-neutralizing antibody response, they did not class-switch to immunoglobulin G class immunoglobulins nor could they maintain long-term virus-neutralizing antibody titers. Eventually, all of the IFN-gamma-deficient mice failed to keep persistent virus in check and developed fatal FV-induced erythroleukemia.