Recombinant Rat Interferon-gamma Research Articles

Effects of Immunosuppressive and Immunostimulative Treatment on Pancreatic Injury and Mortality in Severe Acute Experimental Pancreatitis

Acute pancreatitis is associated with substantial alterations of the immunologic host response which has been claimed to promote remote organ dysfunction, septic complications, and mortality. Treatment with immunomodulating substances has been subject of few experimental studies with still conflicting results. We used the taurocholate-induced model of severe acute pancreatitis (SAP) in rats which were assigned to different treatment regimen: isotonic saline (SAP-S) for nontreated controls, recombinant rat interferon-gamma for immunostimulation (SAP-IFN-gamma), and FK506 for immunosuppression (SAP-FK506). Animals were killed after 3, 6, and 24 hours as well as 3 and 7 days, and parameters of local and systemic severity were assessed. Treatment with IFN-gamma and FK506 attenuated the progression of intrapancreatic necrosis within the first 24 hours after pancreatitis induction along with a substantial reduction of subsequent neutrophil tissue infiltration as shown by decreased myeloperoxidase activity. Enhanced cell death by apoptosis during the postacute course was reduced in FK506-treated animals only. Pancreatic interleukin (IL) 1beta messenger RNA up-regulation occurred early and was slightly suppressed in both treatment groups; tumor necrosis factor alpha (TNF-alpha) and IL-2 messenger RNA expression paralleled the onset of apoptosis and was markedly decreased in IFN-gamma- and FK506-treated rats. The 2 therapeutic regimens had similar effects on intrapancreatic and systemic IL-1beta and TNF-alpha protein release; however, the profiles of both cytokines were differently influenced. Whereas IFN-gamma and FK506 treatment lead to an enhanced intrapancreatic and systemic TNF-alpha protein release during the early course, IL-1beta concentrations were significantly reduced within the late intervals. Seven-day mortality was 44% in saline-, 29% in IFN-gamma-, and 25% in FK506-treated rats (P = not significant). Severe acute pancreatitis is associated with early alterations of the immune response comprising overt T-cell activation and impaired monocyte/macrophage function alike. Targeting either immunologic derangement improves local pancreatic damage and systemic severity. However, because mortality was not improved, a therapeutic benefit of immunomodulating substances in clinical SAP remains to be defined.

Interferon-gamma (IFN-gamma) treatment decreases the inflammatory response in chronic Pseudomonas aeruginosa pneumonia in rats.

In a rat model of chronic Pseudomonas aeruginosa lung infection mimicking cystic fibrosis (CF), we studied whether the inflammatory response could be altered by intraperitoneal treatment with recombinant rat interferon-gamma (rrIFN-gamma). Rats were treated either before or after intratracheal challenge with P. aeruginosa embedded in alginate beads. Rats treated after challenge had a significant reduction in the severity of macroscopic lung inflammation compared with rats treated before challenge (P = 0.004) and controls (P = 0.003). The histopathology in controls was dominated by numerous polymorphonuclear leucocytes (PMN) (> or = 90%) surrounding the alginate beads like in CF. This could be caused by a Th2-like response. In contrast, a complete shift to a chronic-type inflammation dominated by mononuclear leucocytes (> or = 90% lymphocytes and plasma cells) and granulomas was observed in both rrIFN-gamma-treated groups of rats. This could be caused by a Th1-like response. There was no significant difference in lethality between the groups, and the antibody titres against P. aeruginosa sonicate and alginate were similar in the treated rats and controls. Since the ongoing lung tissue damage in CF patients has been shown to be caused by elastase secreted by PMN, which dominate the P. aeruginosa lung infection, our findings offer a possible new strategy of modifying the inflammatory response in CF patients.

Cloning, expression and purification of recombinant cotton rat interferon-gamma.

The hispid cotton rat (Sigmodon hispidus) has proven to be an excellent small animal model; however, immunological studies have been limited due to a lack of available reagents. We report cloning of the cotton rat interferon-gamma (IFN-gamma) cDNA from concanavalin A-stimulated spleen cells using a combination of reverse transcription polymerase amplification reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) protocols. The open reading frame of 513 nucleotides encodes a 170 amino acid (aa) protein followed by a stop codon with a predicted molecular mass of 19548Da. Cotton rat IFN-gamma shares 63, 60, 43 and 43% identity with the hamster, gerbil, mouse and rat counterpart, respectively. IFN-gamma nucleotide sequence corresponding to aa 18-153 was expressed in Escherichia coli under tryptophan promoter control, either fused to a single initiating codon or fused to the thioredoxin coding sequence. Both expression products were found exclusively in bacterial inclusion bodies. Two purification schemes have been developed to purify the product fused to a single methionine. One of them is fast and leads to the recovery of a pure product suitable for use in antibody production. The second protocol, which includes chromatographic steps, allows the use of the purified product for in vitro demonstration of biological activity in a viral cytopathic reduction assay on cotton rat cells.

Semiquantitative measurement of total extracellular matrix protein produced by cultured mesangial cells

SUMMARY: To evaluate the effect of several cytokines on the total production of insoluble ECM, we performed immunoperoxidase staining directly on rat mesangial cells cultured on flat‐bottomed 96‐well plates. the peroxidase activity was demonstrated by orthophenilenediamine (OPD) and measured directly as optic density at 490 nm (OD490) by a microplate reader. After this procedure, cell number in each well was determined by crystal violet staining of which intensity was measured at 540 nm (OD540). the amount of ECM measured as OD490 was corrected by OD540 (OD490/OD540). OD540 was linearly correlated with actual cell number in the well and OD490 for each ECM was firmly correlated with cell number in the well. By this method, dose dependent decrease of fibronectin (FN) and laminin (LM) was observed in the presence of rat recombinant interferon gamma (IFNγ). Human recombinant platelet derived growth factor (PDGF) increased the total production of LM and type IV collagen (Col IV) as well as cell number. This method would also be useful in evaluation for other proteins of insoluble form as well as ECM produced by attached form of cultured cells.

Interferon-gamma-activated primary enterocytes inhibit Toxoplasma gondii replication: a role for intracellular iron.

Toxoplasma gondii is an obligate intracellular parasite that infects a wide variety of nucleated cells in its numerous intermediate hosts including man. The oral route is the natural portal of entry of T. gondii. Ingested organisms are released from cysts or oocysts within the gastrointestinal tract and initially invade the intestinal epithelium. We show that T. gondii invades and proliferates in cultured primary rat enterocytes, obtained with an original procedure. Activation of the enterocytes with rat recombinant interferon-gamma (IFN-gamma) inhibits T. gondii replication, the inhibition being dose dependent. Neither nitrogen and oxygen derivatives nor tryptophan starvation appear to be involved in the inhibition of parasite replication by IFN-gamma. Experiments using Fe2+ salt, carrier and chelator indicate that intracellular T. gondii replication is iron dependent, suggesting that IFN-gamma-treated enterocytes inhibit T. gondii replication by limiting the availability of intracellular iron to the parasite. Our data show that enterocytes probably play a major role on mucosal surfaces as a first line of defence against this coccidia, and possibly other pathogens, through an immune mechanism. The results suggest that limiting the availability of iron could represent a broad antimicrobial mechanism through which the activated enterocytes exert control over intracellular pathogens.

Rat mucosal mast cells: the cultured bone marrow-derived mast cell is biochemically and functionally analogous to its counterpart in vivo.

Mast cells (MC) are biochemically and functionally heterogeneous and the mixture of MC phenotypes varies according to anatomical location. Intestinal mucosal MC (IMMC) have been used to study the mucosal MC subset in the rat, but they are difficult to isolate in sufficient numbers and with consistent purity and viability. Bone marrow-derived MC (BMMC), with an apparent mucosal MC phenotype, can be cultured in large numbers and with high purity from normal rat bone marrow using supernatants from mesenteric lymph node cells of rats infected with the nematode, Nippostrongylus brasiliensis. We have compared serine proteinase content, tumour necrosis factor-alpha (TNF-alpha) storage and secretion, and TNF-alpha-dependent cytotoxicity of IMMC and BMMC to assess the appropriateness of BMMC as in vitro models of mucosal MC. Two-dimensional gel electrophoretic analysis revealed that the overall protein constituents of BMMC and IMMC were highly homologous. Immunoblotting confirmed that both MC types expressed the MMC-associated enzyme, rat mast cell proteinase-2 (RMCP-2), but not RMCP-1, mast cell proteinase-5 (MCP-5) or carboxypeptidase A (CPA), which characterize the connective tissue MC in the rat and which were detected in a representative of this subset, namely, the periotoneal MC (PMC). BMMC demonstrated levels of TNF-alpha-dependent cytotoxicity that were equivalent to those of IMMC. Like IMMC, BMMC contained little stored TNF-alpha, in comparison with PMC, but both MC types generated substantial amounts of TNF-alpha 6 hr following IgE-mediated activation. Pretreatment of PMC with recombinant rat interferon-gamma (IFN-gamma) for 20 hr inhibited anti-immunoglobulin E (anti-IgE)-mediated release of the granule-associated enzyme, beta-hexosaminidase, whereas identically treated BMMC were unresponsive to this cytokine. Similar results have previously been reported for IMMC. Rat BMMC, unlike their more immature and less phenotypically committed counterparts in the mouse, appear therefore to be more appropriate models for studies on the mucosal MC.

Nitric oxide is an effector molecule in inhibition of tumor cell growth by rIFN-gamma-activated rat neutrophils.

This study was designed to determine the effector molecule responsible for the tumor-inhibitory activity of rat neutrophils treated with rat recombinant interferon gamma (rIFN-gamma) in vitro. The results show that nitric oxide (NO) production by neutrophils is dependent on rIFN-gamma concentration, and that neutrophil-mediated tumor cytostasis is in turn dependent on the amount of NO. NO production and tumor cytostasis by rIFN-gamma-activated neutrophils were inhibited completely by N(G) monomethyl-L-arginine (NGMMA), a specific competitive NO production inhibitor. Tumor cytostasis was also inhibited by oxyhemoglobin (HbO(2)), an NO scavenger. An extracellular oxygen radical scavenger, superoxide dismutase (SOD), was found to increase tumor cell inhibition by rIFN-gamma-activated neutrophils by a factor of 4. This SOD-enhanced cytostasis was not even inhibited by catalase. Tumor cytostasis was slightly increased by a hydroxyl radical-(.OH) scavenger, dimethylthiourea (DMTU), which did not affect NO production by rIFN-gamma-activated neutrophils. Our findings suggest that tumor cytostasis of neutrophils activated by rIFN-gamma is mediated by L-arginine-derived nitrogen oxidation products, and that O(2)- produced by these neutrophils reduces NO-mediated tumor cytostasis at low NO concentrations.

Nitric oxide is an effector molecule in inhibition of tumor cell growth by rIFN‐γ‐activated rat neutrophils

This study was designed to determine the effector molecule responsible for the tumor-inhibitory activity of rat neutrophils treated with rat recombinant interferon gamma (rIFN-γ) in vitro. The results show that nitric oxide (NO) production by neutrophils is dependent on rIFN-γ concentration, and that neutrophil-mediated tumor cytostasis is in turn dependent on the amount of NO. NO production and tumor cytostatis by rIFN-γ-activated neutrophils were inhibited completely by NG monomethyl-L-arginine (NGMMA), a specific competitive NO production inhibitor. Tumor cytostasis was also inhibited by oxyhemoglobin (HbO2), an NO scavenger. An extracellular oxygen radical scavenger, superoxide dismutase (SOD), was found to increase tumor cell inhibition by rIFN-γ-activated neutrophils by a factor of 4. This SOD-enhanced cytostasis was not even inhibited by catalase. Tumor cytostasis was slightly increased by a hydroxyl radical-(-OH) scavanger, dimethylthiourea (DMTU), which did not affect NO production by rIFN-γ-activated neutrophils. Our findings suggest that tumor cytostasis of neutrophils activated by rIFN-γ is mediated by L-arginine-derived nitrogen oxidation products, and that O2− produced by these neutrophils reduces NO-mediated tumor cytostasis at low NO concentrations. Int. J. Cancer 71:223-230, 1997. © 1997 Wiley-Liss, Inc.

RIFN-γ-activated rat neutrophils induce tumor cell apoptosis by nitric oxide

We have previously shown that 1) neutrophils activated with various cytokines, including rat recombinant interferon gamma (rIFN-gamma), inhibit tumor cell growth and that 2) nitric oxide (NO) is the effector molecule in tumor inhibition by rIFN-gamma-stimulated rat peritoneal exudate neutrophils. In this study, we examined the nature of tumor cell death induced by rat peritoneal neutrophils activated by rIFN-gamma in order to clarify the mechanism of apoptosis in neoplastic tumor cell death. DNA of 3 syngeneic rat tumor cell lines was significantly fragmented within 3 hr of incubation in the presence of rIFN-gamma-activated neutrophils, and this effect was dependent on both the concentration of rIFN-gamma and the number of neutrophils. This DNA fragmentation was inhibited by L-N-(I-iminoethyl)-ornithine (L-NIO), a NO synthase inhibitor, but not by superoxide dismutase (SOD). Tumor cells treated with the activated neutrophils were shown by electron microscopy to be apoptotic, exhibiting necrotic features with a longer incubation. On the other hand, cytolysis of tumor cells, as determined by a [3H]-uridine release assay, was first observed only at 24 hr of incubation with the rIFN- gamma-activated neutrophils. Taken together, our results suggest that tumor cell apoptosis by activated neutrophils is NO-dependent and that apoptotic tumor cells undergo necrosis as a secondary process. We suggest that tumor cell apoptosis induced by activated neutrophils plays an important role in regulation of neoplastic tumor cell growth and death in vivo.

Interferon gamma down-regulates cytochrome P450 3A genes in primary cultures of well-differentiated rat hepatocytes

Abstract Administration of interferons of both the gamma and alfa/beta classes down-regulates hepatic cytochrome P450 (CYP) genes when administered to humans or rats. In male rats, interferons decrease expression of CYP3A2 at a pretranslational level, but because interferons also release other cytokines in vivo, it is unclear whether this is a direct effect on hepatocytes. We therefore examined the effects of rat recombinant interferon gamma (IFN-gamma) on CYP3A2, other 3A genes, and 2C11 in stable primary cultures of male rat hepatocytes. Hepatocytes were cultured on matrigel in Williams' E, and messenger RNAs (mRNAs) for 3A2, 3A1-like CYPs, and 2C11 mRNA were determined by RNase protection assays. CYP3A and 2C11 proteins were immunoquantified, and their catalytic activities were estimated by testosterone hydroxylation pathways. In control cells, 3A2 mRNA decreased initially but then recovered, and stable levels (15% of freshly isolated cells) were attained between days 3 and 7. Phenobarbital increased 3A2 mRNA to 60-120% values of freshly isolated cells, and mRNA for 3A1-like CYPs were increased 20-fold. In both control and phenobarbital-treated hepatocytes, rat recombinant IFN-gamma (33 U/mL) reduced mRNA for 3A2 and 3A1-like CYPs, as well as 3A protein and testosterone 6 beta-hydroxylase activity. Interferon had no effect on CYP2C11 at mRNA or protein levels in untreated cells, although a reduction in 2C11 protein was evident in phenobarbital-treated cultures. It is concluded that interferon directly alters expression of constitutive and inducible CYP3A genes in well-differentiated male rat hepatocytes in culture, but has no effect on constitutive expression of CYP2C11. (Hepatology 1996 Aug;24(2):367-73)

Interferon ? down-regulates cytochrome P450 3A genes in primary cultures of well-differentiated rat hepatocytes

Administration of interferons of both the gamma and alfa/beta classes down-regulates hepatic cytochrome P450 (CYP) genes when administered to humans or rats. In male rats, interferons decrease expression of CYP3A2 at a pretranslational level, but because interferons also release other cytokines in vivo, it is unclear whether this is a direct effect on hepatocytes. We therefore examined the effects of rat recombinant interferon gamma (IFN-gamma) on CYP3A2, other 3A genes, and 2C11 in stable primary cultures of male rat hepatocytes. Hepatocytes were cultured on matrigel in Williams' E, and messenger RNAs (mRNAs) for 3A2, 3A1-like CYPs, and 2C11 mRNA were determined by RNase protection assays. CYP3A and 2C11 proteins were immunoquantified, and their catalytic activities were estimated by testosterone hydroxylation pathways. In control cells, 3A2 mRNA decreased initially but then recovered, and stable levels (15% of freshly isolated cells) were attained between days 3 and 7. Phenobarbital increased 3A2 mRNA to 60-120% values of freshly isolated cells, and mRNA for 3A1-like CYPs were increased 20-fold. In both control and phenobarbital-treated hepatocytes, rat recombinant IFN-gamma (33 U/mL) reduced mRNA for 3A2 and 3A1-like CYPs, as well as 3A protein and testosterone 6 beta-hydroxylase activity. Interferon had no effect on CYP2C11 at mRNA or protein levels in untreated cells, although a reduction in 2C11 protein was evident in phenobarbital-treated cultures. It is concluded that interferon directly alters expression of constitutive and inducible CYP3A genes in well-differentiated male rat hepatocytes in culture, but has no effect on constitutive expression of CYP2C11.

Interferon gamma increases survival in murine experimental cryptococcosis.

Systemic disease by Cryptococcus neoformans (C. neoformans) is a common opportunistic infection in immunodeficient patients. Cellular immunity seems to be the most important determinant of resistance. The aim of this study was to assess the effect of recombinant rat interferon gamma (IFN-gamma) in murine cryptococcosis (Balb/c mice infected by IP route with the Rivas strain of C. neoformans), evaluating survival time, macroscopic and microscopic examination of the organs, and massive seeding of brain homogenate. IFN-gamma treatment, at a daily dose of 10,000 IU, did not modify significantly these variables when mice were challenged with a high inoculum (10(7) yeasts) and treatment was delayed to 5 days after infection (median survival 21 days in control mice vs. 23 days in IFN-treated). Another set of experiments suggested that IFN-gamma treatment, at a dose of 10,000 IU/day, begun at the moment of infection could be useful (it prolonged survival from 20 to 28 days, although the difference did not achieve statistical signification). When used simultaneously with infection by 3.5 x 10(5) yeasts, IFN-gamma at 10,000 IU/day for 15 days significantly prolonged survival of mice (p = 0.004). These results suggest that, depending on the experimental conditions, IFN-gamma can improve survival of mice infected with a lethal dose of C. neoformans.

Acute and chronic experimental Trypanosoma cruzi infection in the rat. Response to systemic treatment with recombinant rat interferon-gamma.

We examined the effects of recombinant rat interferon-gamma (IFN-gamma) injections on the parasitologic, serologic, immunologic and histopathologic features of acute and chronic experimental Trypanosoma cruzi (T. cruzi) infections in "l" rats. Upon infection at weaning, two rat groups were allocated to receive a 20-day cycle of IFN-gamma injections, 20,000 IU/rat each, which started at 1, and 7 days post-infection (pi). Treatment with IFN-gamma, initiated at either 1 or 7 days pi, resulted in comparatively lower peak parasitemias (P < 0.02) but in similar levels of anti-T. cruzi circulating antibodies and serum IFN-gamma activities. The latter appeared significantly increased during acute infection whereas biologically active tumor necrosis factor was virtually undetectable in serum from infected rats regardless of whether they had been given IFN-gamma or not. The prevalence of chronic focal myocarditis in IFN-gamma-treated infected rats showed no differences with respect to the one recorded in control-infected counterparts. The inverse CD4/CD8 ratio of spleen and lymph node T cells that usually accompanies chronic infection was reversed by IFN-gamma. Mononuclear cells carrying class II I-A and I-E molecules, that were found to have increased at both compartments, appeared also modified upon IFN-gamma treatment with an overincrease of I-A-positive cells, and a normalization of I-E-bearing cells.

The Effect of Interferon-γ 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.

Muramyldipeptide and granulocyte-macrophage colony-stimulating factor enhance interferon-gamma-induced nitric oxide production by rat alveolar macrophages.

Rat alveolar macrophages incubated with recombinant rat interferon-gamma produce L-arginine-dependent nitric oxide, which is rapidly decomposed into nitrite: this production by interferon-gamma was markedly enhanced by granulocyte-macrophage colony-stimulating factor and muramyldipeptide, but not by other cytokines. The enhancement was dependent on the presence of L-arginine in the incubation medium. It was based on a simple synergism between interferon-gamma and muramyldipeptide and a priming effect of granulocyte-macrophage colony-stimulating factor for interferon-gamma-induced nitrite production. These data suggest that cytokine networks are important in the induction of nitric oxide in rat alveolar macrophages.

Inhibition of Rat Bladder Tumor (Rbt323) Growth by Tumor Necrosis Factor Alpha and Interferon-Gamma in Vivo

The antiproliferative activities of recombinant rat gamma-interferon and recombinant human tumor necrosis factor alpha were evaluated in a new rat bladder tumor model, RBT323. The RBT323 is a spontaneously arisen, serially transplantable, bladder tumor in ACI rats. Histologically and ultrastructurally this tumor is a grade 2–3 transitional tumor without squamous cells. The RBT323 appears to be non-immunogenic in tumor excision and rechallenge experiments.The tumor was transplanted subcutaneously and the drugs were administered peritumorally. Gamma-interferon and tumor necrosis factor monotherapy starting two days after tumor implantation showed dose-dependent growth inhibiting effects in the dose range tested. Combination treatment with gamma-interferon and the highest dose of tumor necrosis factor resulted in synergistic antiproliferative effects. After cessation of the combination treatment all tumors resumed growth, initially with a low doubling time, but finally with their original growth rate. Gamma-interferon treatment initiated at a tumor volume of 0.2 to 0.5cm. 3 had no antitumor effects, whereas treatment with 100µg tumor necrosis factor per rat inhibited tumor growth significantly. Different combinations of the cytokines had synergistic effects against established tumors.These studies demonstrate antitumor effects of gamma-interferon and tumor necrosis factor in a rat bladder tumor model system. Combination therapy has synergistic effects and prolongs the time to tumor recurrence. Accepted for publication February 25, 1992.

In vivo administration of recombinant growth hormone or gamma interferon activities macrophages: enhanced resistance to experimental Salmonella typhimurium infection is correlated with generation of reactive oxygen intermediates.

Purified and recombinant forms of growth hormone (GH) as well as of recombinant rat gamma interferon (IFN-gamma) enhance the survival of rats deprived of endogenous pituitary GH secretion by hypophysectomy (HX rats) and infected with virulent Salmonella typhimurium. Macrophages obtained from rats with intact pituitaries (pituitary-intact rats) or HX rats that were treated in vivo with either GH or the closely related hormone prolactin released elevated (P less than 0.05) levels of superoxide anion (O2-) after in vitro opsonized-zymosan stimulation compared with those from placebo-treated animals. These levels of O2- release were similar in magnitude to those of macrophages from rats treated in vivo with IFN-gamma. In time course in vivo macrophage activation studies, both IFN-gamma and GH significantly increased O2- secretion within 24 h, with maximal secretion occurring at day 3. Macrophages obtained from pituitary-intact and HX rats injected in vivo with GH also released elevated (P less than 0.05) levels of hydrogen peroxide (H2O2) and displayed enhanced (P less than 0.01) phagocytic activity toward opsonized Listeria monocytogenes in vitro. The mechanism of action of GH in vivo is likely to be a direct one because resident peritoneal macrophages from rats could be primed in vitro for enhanced secretion of O2- following triggering of these cells with opsonized zymosan. These data show that in vivo administration of two closely related pituitary hormones, GH and prolactin, can effectively prime macrophages, which is consistent with the hypothesis that GH mediates resistance to S. typhimurium by a direct stimulatory action on macrophages.

Modulation of differentiation of rat granulosa cells in vitro by interferon-gamma.

The effects of recombinant rat interferon-gamma (rRaIFN-gamma) and rat IFN (RaIFN, a mixture of IFN-gamma and -alpha) on basal and FSH-induced ovarian granulosa cell function were studied. Granulosa cells were harvested from diethylstilboestrol-treated immature rats and cultured (2 x 10(5) viable cells/well per 0.5 ml) in serum-free medium with or without treatment for 48 h. In the presence of FSH (20 ng/ml), rRaIFN-gamma (10-1000 U/ml) significantly inhibited FSH-stimulated aromatase activity (76.4 +/- 2.3% maximum inhibition compared with FSH treatment alone), inhibin (40.4 +/- 3.7%), progesterone (47.7 +/- 8.6%) and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OHP) (51.8 +/- 1.7%) production in a dose-dependent manner. Furthermore, rRaIFN-gamma inhibited FSH- and forskolin (FSK; 30 mumol/l)-induced extracellular cAMP accumulation (46.0 +/- 6.6% and 29.1 +/- 7.3% respectively). The inhibitory effect of rRaIFN-gamma on FSK-induced cAMP was accompanied by decreased FSK-induced aromatase activity, inhibin, progesterone and 20 alpha-OHP production. rRaIFN-gamma had no detectable effect on aromatase activity, progesterone production and 20 alpha-OHP production in the absence of FSH, but significantly stimulated basal inhibin production by 1.5-fold. rRaIFN-gamma alone also caused a small but significant increase in basal levels of cAMP. The time-course studies showed that FSH-induced aromatase activity and inhibin production were consistently suppressed by rRaIFN-gamma, FSH-induced progesterone and 20 alpha-OHP were inhibited at 1 and 2 days and then stimulated on days 3, 4 and 5 relative to FSH alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcription of class II MHC gene by interferon-gamma in FRTL-5 cells.

The intracellular mechanism by which interferon-gamma induces the expression of class II major histocompatibility complex (MHC) antigen in nonlymphoid cells is not clear. The effect of recombinant rat interferon-gamma (IFN-gamma), and cycloheximide on the expression of class II MHC gene was studied using the techniques of immunocytochemical staining and northern blot analysis. IFN-gamma induced de novo transcription of class II MHC gene and class II MHC antigen expression on the cell surface. Cycloheximide did not inhibit IFN-gamma-induced class II MHC antigen expression in a dose-dependent manner indicating translational blockade. These results suggest that IFN-gamma induces class II MHC antigen expression via de novo transcription of class II MHC gene leading to synthesis of new class II MHC molecule.

The effect of treatment with recombinant γ-interferon on adjuvant-induced arthritis in rats

We have investigated the effects of recombinant rat gamma-interferon (rIFN gamma) on adjuvant-induced arthritis (AA). Lewis rats, inoculated in the left hind-paw with adjuvant (day 0), were given 10(5) U/rat of rIFN gamma daily (days 0 to 20), subcutaneously and intramuscularly on alternate days. rIFN gamma suppressed the secondary phase of swelling of both hind-paw on and after day 18 without influencing the earlier phases, both primary and secondary, of swelling. rIFN gamma also reduced the hind-paw bone lesions, the degree of splenomegaly, and the increase in erythrocyte sedimentation rate and plasma fibrinogen. These results indicate a new aspect of the regulatory role of IFN gamma in chronic inflammation.