Murine IFN-γ Research Articles

Adenovirus-Mediated Murine Interferon-γ Receptor Transfer Enhances the Efficacy of IFN-γ in Vivo

Interferon γ (IFN-γ) plays an important role in immune response, apoptosis, and anti-tumor activity. Its biological activity depends on expression of IFN-γ receptor (IFN-γR). To address whether increased expression of IFN-γR is associated in vivo with a higher biological response by IFN-γ, we constructed an adenovirus vector including murine IFN-γR (Ad-mIFN-γR). We confirmed the appropriate function of mIFN-γR derived from Ad-mIFN-γR based on the observation of signal transduction and transcription. We also found that elevated expression of mIFN-γR increases sensitivity to recombinant murine IFN-γ (rmIFN-γ) in vitro in target cells. Furthermore, we demonstrated that the growth rate of tumors transfected with Ad-mIFN-γR is suppressed in response to rmIFN-γ in vivo and that such growth suppression is partly due to apoptosis. To our knowledge, this is the first report of adenovirus-mediated IFN-γR gene transfer being effective in augmenting the biological activity of IFN-γ, and the strategy employed in the present study will be useful in studying other kinds of cytokine receptors and applications to gene therapy for cancer and infectious diseases.

Interferon-gamma-mediated activation and ubiquitin-proteasome-dependent degradation of PPARgamma in adipocytes.

Interferon-gamma (IFNgamma) treatment of adipocytes results in a down-regulation of the peroxisome proliferator-activated receptor gamma (PPARgamma). The decrease in PPARgamma expression is mediated by inhibition of PPARgamma synthesis and increased degradation of PPARgamma. In this study, we demonstrate that both PPARgamma1 and PPARgamma2 are targeted to the proteasome under basal conditions and that PPARgamma1 is more labile than PPARgamma2. The IFNgamma-induced increase in PPARgamma turnover is blocked by proteasome inhibition and is accompanied by an increase in PPARgamma-polyubiquitin conjugates. In addition, IFNgamma treatment results in the transcriptional activation of PPARgamma. Similar to ligand-dependent activation of PPARgamma, IFNgamma-induced activation was greater in the phosphorylation-deficient S112A form of PPARgamma when compared with wild-type PPARgamma. Moreover, the inhibition of ERKs 1 and 2 with a MEK inhibitor, U1026, lead to an inhibition in the decay of PPARgamma proteins, indicating that serine phosphorylation influences the degradation of PPARgamma in fat cells. Our results also demonstrate that the proteasome-dependent degradation of PPARgamma does not require nuclear export. Taken together, these results indicate that PPARgamma is targeted to the ubiquitin-proteasome pathway for degradation under basal conditions and that IFNgamma leads to an increased targeting of PPARgamma to the ubiquitin-proteasome system in a process that is affected by ERK-regulated serine phosphorylation of PPARgamma proteins.

Intratumoral delivery of an interferon gamma retrovirus-producing cells inhibits growth of a murine melanoma by a non-immune mechanism

Interferon gamma (IFNγ) is a potent inhibitor of cell growth effective against a wide range of tumor-derived cell lines. We cloned murine IFNγ cDNA into a retroviral vector and created a packaging cell line (Am-gamma) producing this IFNγ-encoding retrovirus. In a pilot experiment, admixing and co-injection of equal numbers of retrovirus-producing and target B16 melanoma cells led to high rates of infection and strong suppression of neoplastic growth. This effect was observed in the absence of measurable systemic production of IFNγ and could be reproduced in animals lacking cytotoxic immune responses. Tumor angiogenesis was unaffected and no increase in apoptosis was apparent; however, mitotic indices were greatly reduced in Am-gamma-containing abortive tumors. We thus concluded that IFNγ directly affects proliferation of B16 cells. Indeed, exposure of B16 cells to IFNγ in vitro inhibits cell division, as measured by a thymidine incorporation assay. Most importantly, repeated intratumoral injections of Am-gamma stunted growth of established B16 melanomas in 75% of treated animals. Thus, this approach can serve as a prototype for new anti-cancer modalities.

Neutralization of interleukin-18 reduces severity in murine colitis and intestinal IFN-gamma and TNF-alpha production.

Interleukin (IL)-18, initially described as interferon (IFN)-gamma-inducing factor, is expressed in the inflamed mucosa of patients with Crohn's disease. To investigate the role of IL-18 in intestinal inflammation, the effect of neutralizing antimurine IL-18 antiserum in dextran sulfate sodium (DSS)-induced colitis in BALB/c and C57BL/6 mice was examined. During a dose response of DSS, levels of colonic IL-18 increased parallel with clinical worsening. With the use of confocal laser microscopy, the increased IL-18 was localized to the intestinal epithelial layer. Anti-IL-18 treatment resulted in a dose-dependent reduction of the severity of colitis in both BALB/c and C57BL/6 mice. Colon shortening following DSS-induced colitis was partially prevented in the treatment groups. In the colon tissue homogenates, IFN-gamma concentrations were lower in the anti-IL-18-treated DSS-fed mice compared with untreated DSS-fed mice. This suppressive effect of anti-IL-18 administered in vivo was also observed on spontaneous tumor necrosis factor-alpha, IL-18, and IFN-gamma production from ex vivo colon organ cultures. The stimulation of lamina propria mononuclear cells by IL-18 and IL-12 resulted in a synergistic increase in IFN-gamma synthesis. These findings suggest that IL-18 is a pivotal mediator in experimental colitis.

The many faces of host responses to tuberculosis.

Tuberculosis remains today one of the top three fatal infectious diseases, together with acquired immune deficiency syndrome (AIDS) and malaria. During the last decade, 90 million new infections occurred, resulting in approximately 30 million deaths. Although there is currently effective chemotherapy, consisting of three specific drugs, this regimen must be continued for a period of at least 6 months, which in many cases, results in problems with compliance. Lack of compliance further impacts on the development of multidrug-resistant strains of the bacterium, which consequently raises the cost of treatment, making the expense of curing tuberculosis prohibitive in many developing countries. Despite the enormous numbers of people infected with this organism, it is estimated that only 10% of affected individuals show evidence of clinical symptoms. Many parameters, notably socio-economic factors, co-infection with human immunodeficiency virus (HIV) and genetic predisposition of the host, influence the susceptibility to disease. Much work has been invested to elucidate the biology of the interaction between Mycobacterium tuberculosis and its host, both in experimental animal models and in clinical studies. Here we review some of the latest developments in the understanding of the immune response required to control this pathogen. It is hoped that further progress in this field will lead to a more rational approach towards the development of an effective vaccine and novel chemotherapeutic agents.

Immunoadjuvant activity of interferon-γ-liposomes co-administered with influenza vaccines

In an attempt to potentiate the relatively low immunogenicity of the currently used influenza vaccines, especially in high-risk groups, monovalent and divalent subunit vaccine preparations were co-administered with free or liposome-associated murine interferon γ (mIFNγ) as an adjuvant. Recombinant murine IFNγ was entrapped (50–70% efficiency) in two types of large multilamellar vesicles: mIFNγ-LIP A-‘conventional’ liposomes, and mIFNγ-LIP B- ‘surface-depleted’ liposomes, in which 60 and 8% of the associated cytokine was located at the external liposome membrane, respectively. Subunit preparations containing the viral surface proteins hemagglutinin and neuraminidase (HN) were injected once, i.p. (0.5 μg each), into BALB/c mice, alone and combined with free or liposomal mIFNγ (mIFNγ-LIP, 0.5 or 3.0 μg). Sera were tested 3–16 weeks post-vaccination by hemagglutination inhibition (HI), and by ELISA for IgG1 and IgG2a antibodies (Abs). In addition, protective immunity against intranasal viral infection was assayed at 11 and 17 weeks post-vaccination. The results showed that: (a) Vaccination with HN alone produces very low HI and IgG titers and does not afford any protection. (b) Although co-administration with free mIFNγ (particularly using 3.0 μg) markedly enhances HI titer as well as the IgG1 and IgG2a levels, protection is negligible (0–33%). (c) In most cases, mIFNγ-LIP is significantly more potent than free mIFNγ (2–40-fold increase in Ab titer), and the low dose (0.5 μg) is generally more efficient than the high dose. Up to 83% of the mice co-vaccinated with mIFNγ-LIP were protected against viral challenge. (d) Both the IgG2a level and the HI titer appear to be crucial for protection. (e) Although the two liposomal preparations differ in their cytokine release profile in vivo and in their bioactivity in vitro, their adjuvant activity is comparable.

Vaccinia virus vectors with an inactivated gamma interferon receptor homolog gene (B8R) are attenuated In vivo without a concomitant reduction in immunogenicity.

The vaccinia virus (VV) B8R gene encodes a secreted protein with homology to the gamma interferon (IFN-gamma) receptor. In vitro, the B8R protein binds to and neutralizes the antiviral activity of several species of IFN-gamma, including human and rat IFN-gamma; it does not, however, bind significantly to murine IFN-gamma. Here we report on the construction and characterization of recombinant VVs (rVVs) lacking the B8R gene. While the deletion of this gene had no effect on virus replication in vitro, rVVs lacking the B8R gene were attenuated for mice. There was a significant decrease in weight loss and mortality in normal mice, and nude mice survived significantly longer than did controls inoculated with parental virus. This is a surprising result considering the minimal binding of the B8R protein to murine IFN-gamma and its failure to block the antiviral activity of this cytokine in vitro. Such reduction in virulence could not be determined in rats, since they are considerably more resistant to VV infection than are mice. Finally, deletion of the B8R gene had no detectable effects on humoral immune responses. Mice and rats vaccinated with the rVVs showed identical humoral responses to both homologous and heterologous genes expressed by VV. This study demonstrates that the deletion of the VV B8R gene leads to enhanced safety without a concomitant reduction in immunogenicity.

Aerosol infection of mice with recombinant BCG secreting murine IFN-γ partially reconstitutes local protective immunity

To better understand the contribution of interferon-gamma (IFN-γ) to the immune response during the first 60 days of mycobacterial infection in the lungs, IFN-γ gene disrupted (IFN-γ-/-) mice were infected via aerosol with recombinant Mycobacterium bovis Bacillus Calmette-Guerin (BCG) secreting murine IFN-γ (BCG-IFN-γ) and compared to mice infected with recombinant BCG containing the vector only (BCG-vector). When IFN-γ-/- mice were infected with BCG-vector, increasing bacillary loads and large undifferentiated granulomas that did not express inducible nitric oxide synthase (iNOS) were observed in the lungs. In contrast, infection with BCG-IFN-γ resulted in reduced bacillary load and better differentiated granulomas containing epithelioid macrophages expressing iNOS as well as reduced levels of interleukin 10 (IL-10) mRNA. However, local production of IFN-γ by the recombinant BCG did not protect IFN-γ-/- mice from subsequent challenge with M. tuberculosis. Infection of IFN-γ-/- peritoneal macrophages in vitro with BCG-IFN-γ led to induction of iNOS expression and lower IL-10 mRNA levels. Nevertheless, the growth of the intracellular BCG was unaffected. Since IFN-γ induced-iNOS protein and reduced IL-10 production were insufficient to control mycobacterial growth in vitro, the results suggest that additional mediator(s) present in vivo are required for control of mycobacterial growth.

Interferon gamma contributes to initiation of uterine vascular modification, decidual integrity, and uterine natural killer cell maturation during normal murine pregnancy.

The dominant lymphocytes in human and murine implantation sites are transient, pregnancy-associated uterine natural killer (uNK) cells. These cells are a major source of interferon (IFN)-gamma. Implantation sites in mice lacking uNK cells (alymphoid recombinase activating gene [RAG]-2(-/)- common cytokine receptor chain gamma [gamma(c)](-/)-) or IFN-gamma signaling (IFN-gamma(-/)- or IFN-gammaRalpha(-/)-) fail to initiate normal pregnancy-induced modification of decidual arteries and display hypocellularity or necrosis of decidua. To investigate the functions of uNK cell-derived IFN-gamma during pregnancy, RAG-2(-/)-gamma(c)(-/)- females were engrafted with bone marrow from IFN-gamma(-/)- mice, IFN-gamma signal-disrupted mice (IFN-gammaRalpha(-/)- or signal transducer and activator of transcription [Stat]-1(-/)-), or from mice able to establish normal uNK cells (severe combined immunodeficient [SCID] or C57BL/6). Mated recipients were analyzed at midgestation. All grafts established uNK cells. Grafts from IFN-gamma(-/)- mice did not reverse host vascular or decidual pathology. Grafts from all other donors promoted modification of decidual arteries and decidual cellularity. Grafts from IFN-gammaRalpha(-/)- or Stat-1(-/)- mice overproduced uNK cells, all of which were immature. Grafts from IFN-gamma(-/)-, SCID, or C57BL/6 mice produced normal, mature uNK cells. Administration of murine recombinant IFN-gamma to pregnant RAG-2(-/)-gamma(c)(-/)- mice initiated decidual vessel modification and promoted decidual cellularity in the absence of uNK cells. These in vivo findings strongly suggest that uNK cell-derived IFN-gamma modifies the expression of genes in the uterine vasculature and stroma, which initiates vessel instability and facilitates pregnancy-induced remodeling of decidual arteries.

Transgenic mice expressing surface markers for IFN-γ and IL-4 producing cells

The detection of T H1-type and T H2-type cells directly in situ would be of great value in the study of T H development and function in vivo. Transgenic mice expressing human Thy1 and mouse Thy1.1 under the control of the murine IFN-γ and IL-4 promoters, respectively, have been generated. The hThy1 + cells represent (with some temporal lag) most of the IFN-γ-producing CD4 + T-cells, while the mThy1.1 + cells represent only a percentage of IL-4 secreting cells. This may be due to mono-allelic expression of the IFN-γ and IL-4 genes. Since permeabilization is not required for the detection of the transgenic surface markers, these transgenic mice can facilitate the detection of T H1-type and T H2-type cells by flow cytometry with surface immunofluorescent staining. These surface markers should permit isolation of viable cells according to their T H type for adoptive transfer experiments, and may serve as a model system for tracing the development of T H1 and T H2-type cells in vivo.

Cutting edge: IL-15 costimulates the generalized Shwartzman reaction and innate immune IFN-gamma production in vivo.

Sequential administration of LPS to SCID mice results in the generalized Shwartzman reaction, manifesting as rapid mortality via cytokine-induced shock. Here we demonstrate that in vivo neutralization of IL-15 before LPS priming significantly reduced lethality in this reaction (p = 0.0172). We hypothesize that LPS priming induces IL-12 and IL-15 that costimulate NK cell-derived IFN-gamma. Such IFN-gamma may then in turn sensitize macrophages to elicit the Shwartzman reaction following a subsequent LPS challenge. Supporting this, IL-12 and IL-15 synergized to induce murine NK cell IFN-gamma production in vitro. LPS stimulation of SCID mouse splenocytes resulted in measurable IFN-gamma production, which was reduced when IL-15 was neutralized or IL-2/15Rbeta was blocked. Pretreatment with either anti-IL-2/15Rbeta or anti-IL-15 Abs reduced serum IFN-gamma protein following LPS administration to SCID mice. Collectively, these data provide the first in vivo evidence that IL-15 participates in LPS-induced innate immune IFN-gamma production and significantly contributes to the lethal Shwartzman reaction.

Interleukin-1 inhibits gamma interferon-induced bacteriostasis in human uroepithelial cells.

The most prominent gamma interferon (IFN-gamma)-induced antimicrobial effector mechanisms are the induction of nitric oxide (NO) synthase (NOS) and of indoleamine 2,3-dioxygenase (IDO) activity. We have recently found that human glioblastoma cells and human macrophages inhibit the growth of group B streptococci after stimulation with IFN-gamma. In this report, we show that in addition, human RT4 (uroepithelial) cells can inhibit the growth of enterococci. Murine macrophages (RAW cells) are unable to inhibit bacterial growth after IFN-gamma stimulation. Stimulation of human glioblastoma cells, macrophages, and RT4 cells with human IFN-gamma results in a strong expression of IDO activity; however, NO production remains undetectable. In strong contrast, murine RAW cells produce large amounts of NO when stimulated with murine IFN-gamma and IDO activity is not detectable. Interleukin-1 (IL-1) induces NO synthase in human RT4 cells when the cells are costimulated with IFN-gamma. We found that IL-1 inhibits IFN-gamma-stimulated IDO activity and antimicrobial effects in RT4 cells, while in human glioblastoma cells, which lack detectable NO synthase activity, neither of these effects was altered by costimulation with IFN-gamma and IL-1. The IL-1-mediated inhibition of IDO activity and of subsequent antibacterial effect is due to the production of NO. This conclusion was supported by evidence that N(G)-monomethyl-L-arginine, a competitive inhibitor of inducible NOS activity, is able to block the inhibitory action of IL-1 on IFN-gamma-induced bacteriostasis. We therefore conclude that NO production does not inhibit the growth of enterococci but might be involved in the regulation of IDO activity in some human cells.

CD4+T cells eliminate MHC class II-negative cancer cellsin vivoby indirect effects of IFN-γ

CD4(+) T cells can eliminate tumor cells in vivo in the absence of CD8(+) T cells. We have CD4(+) T cells specific for a MHC class II-restricted, tumor-specific peptide derived from a mutant ribosomal protein expressed by the UV light-induced tumor 6132A-PRO. By using neutralizing mAb specific for murine IFN-gamma and adoptive transfer of CD4(+) T cells into severe combined immunodeficient mice, we show that anti-IFN-gamma treatment abolishes the CD4(+) T cell-mediated rejection of the tumor cells in vivo. The tumor cells were MHC class II negative, and IFN-gamma did not induce MHC class II expression in vitro. Therefore, the tumor-specific antigenic peptide must be presented by host cells and not the tumor cells. Tumor cells transduced to secrete IFN-gamma had a markedly reduced growth rate in severe combined immunodeficient mice, but IFN-gamma did not inhibit the growth of the tumor cells in vitro. Furthermore, tumor cells stably expressing a dominant-negative truncated form of the murine IFN-gamma receptor alpha chain, and therefore insensitive to IFN-gamma, nevertheless were rejected by the adoptively transferred CD4(+) T cells. Thus, host cells, and not tumor cells, seem to be the target of IFN-gamma. Together, these results show that CD4(+) T cells can eliminate IFN-gamma-insensitive, MHC class II-negative cancer cells by an indirect mechanism that depends on IFN-gamma.

Expression of interferon-γ by stromal cells inhibits murine long-term repopulating hematopoietic stem cell activity

Several lines of evidence suggest that overexpression of interferon γ (IFN-γ) in the marrow microenvironment may play a role in the pathogenesis of marrow suppression in aplastic anemia. We previously showed that overexpression of IFN-γ by marrow stromal cells inhibits human long-term culture initiating cell activity assayed in vitro to a much greater degree than the addition of soluble IFN-γ. The effect of IFN-γ on true repopulating stem cells assayed in vivo has not been studied previously. We compared the effect of co-culture of murine marrow cells in the presence of stromal cells transduced with a retroviral vector expressing murine IFN-γ vs stromal cells transduced with a control neo vector. Using a murine congenic competitive repopulation assay, there was significantly less long-term repopulating stem cell activity remaining after culture on mIFN-γ–expressing stroma as compared to control stroma. We also investigated the effect of directly transducing murine bone marrow cells with the mIFN-γ or control vector. Marrow cells transduced with either vector were transplanted into W/W v recipient mice. The percentage of vector-containing cells in the mIFN-γ mice was significantly lower than in the control mice, suggesting that mIFN-γ–transduced primitive cells may not have survived culture, or that mIFN-γ directly decreases gene transfer into repopulating cells. Despite no significant differences in white or red blood cells in the mice transplanted with the mIFN-γ–transduced cells, the number of bone marrow colony-forming unit-C 16 weeks after transplantation was significantly lower in the IFN-γ group. These data indicate that ectopic or overexpression of mIFN-γ, especially by marrow microenvironmental elements, may have a marked effect on primitive hematopoiesis as assayed in vivo. Published by Elsevier Science Inc.

IFN-γ and CD8+ T Cells Restore Host Defenses AgainstPneumocystis cariniiin Mice Depleted of CD4+ T Cells

AbstractHost defenses against infection are profoundly compromised in HIV-infected hosts due to progressive depletion of CD4+ T lymphocytes and defective cell-mediated immunity. Although recent advances in antiretroviral therapy can dramatically lower HIV viral load, blood CD4+ T lymphocytes are not restored to normal levels. Therefore, we investigated mechanisms of host defense other than those involving CD4+ T lymphocytes against a common HIV-related opportunistic infection, Pneumocystis carinii (PC) pneumonia. Using CD4-depleted mice, which are permissive for chronic PC infection, we show that up-regulation of murine IFN-γ by gene transfer into the lung tissue results in clearance of PC from the lungs in the absence of CD4+ lymphocytes. This resolution of infection was associated with a >4-fold increase in recruited CD8+ T lymphocytes and NK cells into the lungs. The role of CD8+ T cells as effector cells in this model was further confirmed by a lack of an effect of IFN-γ gene transfer in scid mice or mice depleted of both CD4+ and CD8+ T cells. Cytokine mRNA analysis revealed that recruited, lung-derived CD8+ T cells had greater expression of IFN-γ message in animals treated with the IFN-γ gene. These results indicate that CD8+ T cells are capable of clearing PC pneumonia in the absence of CD4+ T cells and that this host defense function of CD8+ T cells, as well as their cytokine repertoire, can be up-regulated through cytokine gene transfer.

Unrestricted Agonist Activity on Murine and Human Cells of a Lipopeptide Derived from IFN-γ

We describe the isolation of a synthetic agonist of IFN-γ (L-mIFN-γ-Ct) active on mouse and human cells. Its biological activity is the result of the ability of the C-terminal extremity of murine IFN-γ to interact with the intracellular part of IFNγ-R and the observation that the modification of peptides by a palmitic acid enables their cytoplasmic delivery. L-mIFN-γ-Ctstimulated murine cells exhibited an increase in MHC class II molecules and FcγRII/III expression and conferred protection against viral lysis. Unresponsiveness to L-mIFN-γ-Ctof cells recovered from IFNγ-R α-chain knockout mice indicated the involvement of IFNγ-R in the biological activities observed. Induction of VCAM-1, ICAM-1, and HLA-DR expression on human cells stimulated with L-mIFN-γ-Ctdemonstrated an abrogation of species specificity. These results describe the development of a new synthetic agonist of IFN-γ, which substitutes for the native cytokine in any IFN-γ responsive cells, by acting intracellularly on IFN-γR.

Immunomodulation of murine experimental SLE-like disease by interferon-gamma.

The objective of this study was to assess the impact of murine recombinant IFN-gamma and anti-IFN-gamma monoclonal antibody on the BALB/c mice experimental model of lupus. BALB/c female mice were immunized with a human anti-DNA antibody that carries the 16/6 idiotype. These mice were divided into several therapeutic groups according to different treatment strategies; injection with mouse recombinant IFN-gamma, anti-IFN-gamma mAb, phosphate-buffered saline (PBS), irrelevant mouse IgG and control groups that were neither treated nor immunized with the human anti-DNA antibody. The administration of IFN-gamma, intensified the degree of clinical, histological and serological parameters in this model of BALB/c murine lupus. This immunomanipulation decreased the mice longevity. All the laboratory parameters reflected acceleration of the disease in the IFN-gamma treated group as an elevated sedimentation rate, decreased white blood cell count and the development of massive proteinuria. One month after the boost injection, all the mice that were immunized with the anti-DNA antibody, developed high titers of autoantibodies; however, following an additional month, their levels declined in the IFN-gamma treated group. These findings were in concordance with an increased glomerular deposition of immune complexes in the IFN-gamma treated mice. IFN-gamma upregulated the levels of IL-4 and increased the number of IL-4 and IL-6 secreting splenocytes. In conclusion IFN-gamma administration can aggravate the clinical and laboratory outcome of 16/6 id induced lupus in BALB/c mice.

Expression and Regulation of Interferon- -Inducible Protein 10 Gene in Rat Leydig Cells

In the present study, we report the cloning of a gene that is differentially expressed in normal adult rat Leydig cells and whose expression is inhibited by hCG but is induced by interferon-γ (IFNγ). DNA sequence analysis identified this gene as rat IFNγ-inducible protein 10 (IP-10), a member of the -C-X-C- chemokine superfamily of proinflammatory cytokines. High levels of IP-10 messenger RNA (mRNA) were constitutively expressed in freshly isolated and primary cultured Leydig cells. hCG inhibited this expression in a dose-dependent manner. The addition of 1 ng/ml hCG inhibited IP-10 mRNA levels more than 80%. Conversely, IP-10 mRNA levels were markedly increased in response to murine interleukin-1α, murine tumor necrosis factor-α, and murine IFNγ by 3.3-, 10-, and 26-fold, respectively. Concomitant addition of murine interleukin-1α, murine tumor necrosis factor-α, and murine IFNγ synergistically increased IP-10 mRNA levels by 58-fold. Furthermore, in addition to one previously described rat IP-10 mRNA transcript (1.5 kb), another larger transcript (2.7 kb) was identified by Northern blot in rat Leydig cells. After screening a rat testis complementary DNA library, we obtained a partial structural gene and an intron sequence, which possibly originated from the larger transcript of rat IP-10 mRNA. Histochemical and immunocytochemical staining revealed that purified cells were positive for 3β-hydroxysteroid dehydrogenase and IP-10, confirming that IP-10 is indeed present in Leydig cells. IP-10 antisense oligonucleotides enhanced basal and hCG-induced testosterone formation. This suggests that endogenous IP-10 has an inhibitory effect on Leydig cell steroidogenesis. In conclusion, IP-10 is expressed in rat Leydig cells and may have paracrine and autocrine effects on testicular function.

Blockade of Endogenous TNF-α Exacerbates Primary and Secondary Pulmonary Histoplasmosis by Differential Mechanisms

AbstractWe investigated the mechanisms by which endogenous TNF-α modulates host defenses during experimental primary and secondary pulmonary infection with Histoplasma capsulatum (Hc). Neutralization of TNF-α in vivo resulted in increased CFU and 100% mortality in naive and immune mice challenged with Hc intranasally. Levels of IFN-γ and granulocyte macrophage-CSF were elevated in TNF-α-neutralized naive mice, whereas IL-4, -6, -10 and TGF-β did not differ from controls. In contrast, in secondary histoplasmosis, significant elevations of IL-4 and -10 were observed in TNF-α-depleted mice. Alveolar macrophages (Mφ) did not exert fungistatic activity against Hc after exposure to recombinant murine TNF-α, recombinant murine IFN-γ, or both. The increase in susceptibility to primary Hc infection was associated with diminished production of reactive nitrogen intermediates by alveolar Mφ from TNF-α-depleted mice, whereas production of nitric oxide during secondary histoplasmosis was similar in both groups. Upon secondary challenge, TNF-α-depleted mice were rescued by concomitant neutralization of IL-4 and IL-10, but not either cytokine alone. Thus, TNF-α is critical for controlling primary and secondary infection with Hc, and the mechanisms that lead mice to succumb to primary or secondary infection when endogenous TNF-α is blocked are different.

Transient Gene Transfer of IL-12 Regulates Chemokine Expression and Disease Severity in Experimental Arthritis

Murine collagen-induced arthritis (CIA) is characterized by pannus formation, cell infiltration, and cartilage erosion, and shares histologic and immunologic features with rheumatoid arthritis. Numerous cytokines are reportedly associated with RA and/or CIA; however, their mechanistic role is not clear. To determine the role of IL-12 in CIA, DBA/1 LacJ mice were administered 3 x 10(8) plaque-forming units of mIL-12 i.p. in a nonreplicating adenoviral vector (AdIL-12) on day 25 following primary type II collagen immunization. Our studies demonstrated that systemic transient overexpression of IL-12 accelerated disease progression and augmented the arthritis severity relative to mice expressing a replication-deficient, E1-deleted Ad5 construct. A likely mechanism for this increase in pathology was the increase in the expression of cytokines and chemokines known to play a proinflammatory role in disease. In particular, levels of murine IFN-gamma were significantly increased in mice overexpressing AdIL-12 relative to the replication-deficient, E1-deleted Ad5 construct. Interestingly, the C-X-C chemokine murine macrophage inflammatory protein-2, as well as the C-C chemokines murine monocyte chemoattractant protein-1 and murine macrophage inflammatory protein-1alpha were up-regulated by AdIL-12 relative to controls. In an additional set of studies, neutralization of endogenous IL-12 in CIA mice was shown to delay disease onset and attenuate disease severity. IFN-gamma levels in the mice receiving anti-IL-12 were significantly decreased in joint homogenates. These studies demonstrate that IL-12 is an important cytokine involved in controlling the production of chemokines/cytokines leading to the evolution of experimental arthritis.