ABSTRACT

Treatment of three β-human chorionic gonadotrophin (β-hCG)-expressing bladder tumour cell lines with interferon-α (IFN-α) (5000 U/per 10⁶ cells) enhanced the rate of β-hCG secretion from 34·2 ±0·9 to 102·5 ± 0·1 mIU/10⁶ cells per 72 h in cell line 5637; 111·15 ± 11·75 to 261·8± 51·75 mIU/10⁶ cells per 72 h in cell line RT112 and 503·25 ± 28·55 to 1361·65± 110·3 mIU/10⁶ cells per 72 h in cell line SCaBER. IFN-γ had no effect on the rate of β-hCG secretion. Both interferons reduced the growth rate of the cells: incorporation of radiolabelled thymidine was reduced by 15–45% in the presence of IFN-α and by 20–53% with IFN-γ. Enhancement of β-hCG secretion by IFN-α was dose-dependent over the range 5–50 000 U/10⁶ cells. Analysis of cell cycle profiles by flow cytometry showed no increase in the proportion of cells in the G₀G₁ phase in cultures treated with IFN-α.

The conceptus of some species produces substances which are either luteotrophic or anti-luteolytic. In sheep, the corpus luteum is maintained by ovine trophoblast protein-I, which has been shown to have structural homology with human IFN-α. In primates and a few other higher mammals, early pregnancy is maintained by chorionic gonadotrophin. IFN-α is also an early product of the human conceptus. We have now shown that IFN-α enhances the ectopic production of the β-subunit of hCG by bladder tumour cells. This study suggests a direct transcription/translational effect of this cytokine on the expression of a reproductive endocrine gene.

Journal of Endocrinology (1989) 123, 501–507

ABSTRACT

Ovine trophoblastin (oTP) is a natural interferon of the class-II interferon-α subfamily. Recombinant ovine trophoblastin (r.oTP), produced by genetic engineering, was purified by anion-exchange HPLC. The product exhibited a high degree of homogeneity (>98%), and similar immunological cross reaction and antiviral activity to natural oTP. Antiluteolytic activity of r.oTP was established by intrauterine injection in two groups of cyclic recipient ewes. Control group A included 10 ewes which received sterile BSA in saline twice daily for 8 days (from day 10-12 of oestrous cycle). Experimental group B included 17 ewes which received 80 μg (4 ewes), 170 μg (8 ewes) or 340 μg (5 ewes) r.oTP daily for 8 days. Maintenance of functional corpora lutea for 1 month or more was observed in 4 out of 5 ewes which received high doses of r.oTP. These results indicate that oTP alone extends luteal secretory activity.

ABSTRACT

The effects of recombinant rat interferon-γ (rRaIFN-γ) and rat IFN (RaIFN, a mixture of IFN-γ and -α) on basal and FSH-induced ovarian granulosa cell function were studied. Granulosa cells were harvested from diethylstilboestrol-treated immature rats and cultured (2 × 10⁵ viable cells/well per 0·5 ml) in serumfree medium with or without treatment for 48 h. In the presence of FSH (20 ng/ml), rRaIFN-γ (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 20α-hydroxypregn-4-en-3-one (20α-OHP) (51·8±1·7%) production in a dose-dependent manner. Furthermore, rRaIFN-γ inhibited FSH- and forskolin (FSK; 30 μmol/l)-induced extracellular cAMP accumulation (46·0 ± 6·6% and 29·1 ± 7·3% respectively). The inhibitory effect of rRaIFN-γ on FSK-induced cAMP was accompanied by decreased FSK-induced aromatase activity, inhibin, progesterone and 20α-OHP production. rRaIFN-γ had no detectable effect on aromatase activity, progesterone production and 20α-OHP production in the absence of FSH, but significantly stimulated basal inhibin production by 1·5-fold. rRaIFN-γ alone also caused a small but significant increase in basal levels of cAMP. The timecourse studies showed that FSH-induced aromatase activity and inhibin production were consistently suppressed by rRaIFN-γ, FSH-induced progesterone and 20α-OHP were inhibited at 1 and 2 days and then stimulated on days 3, 4 and 5 relative to FSH alone. There was no difference in DNA content between treatment and non-treatment wells during 5 days of culture. RaIFN had similar effects to rRaIFN-γ. We conclude that IFN-γ can inhibit FSH-induced granulosa cell differentiation and that, in the absence of FSH, IFN-γ stimulated undifferentiated granulosa cells to produce more inhibin. The mechanism of its action is likely to involve changes in cAMP production.

Journal of Endocrinology (1992) 133, 131–139

Abstract

Renal cell carcinoma is a chemotherapy-resistant tumor which is relatively responsive to immunotherapy. Immunotherapeutic regimes employ interferons or interleukin 2 with or without lymphokine-activated killer cells. Secondary cytokines, induced by interleukin 2 or interferon, may have an important impact on their anti-neoplastic activity. Notable among them is tumor necrosis factor (TNFα). We assessed the effect of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) on the susceptibility of the human renal cell carcinoma cell line SK-RC-29 to the cytotoxic and cytostatic actions of TNFα, interferon a and lymphokine-activated killer cells. Using uptake of the vital dye neutral red as an indicator of viable cell number, we found that addition of 1,25(OH)₂D₃ (100 nm) to TNFα (30 ng/ml)-treated cultures resulted in a 2·6 ± 0·2-fold (mean ± s.e.) increase in the cytotoxic effect of the cytokine. The potentiating effect of 1,25(OH)₂D₃ was dose-dependent, and significant at concentrations equal to or higher than 10 nm. Another dihydroxylated vitamin D metabolite, 24,25(OH)₂D₃, had no effect on TNFα action. The cytotoxic effect of TNFα increased whereas the potentiation by 1,25(OH)₂D₃ decreased with cell density in culture. 1,25(OH)₂D₃, in contrast to its potentiating effect on TNFα action, did not modulate the cytostatic effect of interferon α or the susceptibility of SK-RC-29 to killing by lymphokine-activated killer cells. The findings reported here may explain some of the in vivo anti-tumor activity of 1,25(OH)₂D₃ and provide a rationale for the employment of active vitamin D analogs during immune anti-cancer therapy.

Journal of Endocrinology (1996) 149, 327–333

Interferon stimulated gene 17 (ISG17) and Mx are up-regulated in the ruminant uterus in response to interferon-tau (IFNtau) during early pregnancy. Recent evidence strongly indicates that expression of ISGs occur only in stroma (ST) and glandular epithelium (GE) during this time as a result of transcriptional repression by interferon regulatory factor two (IRF-2) expression in the LE. The present report tested this hypothesis by examining mRNA and protein expression of ISG17 and Mx in serial uterine cross-sections obtained from cyclic and early pregnant ewes. In situ and immunocytochemical analysis revealed that ISG17 mRNA and protein were low to undetectable, whereas Mx mRNA was expressed in the lumenal (LE) and superficial GE at all days of the estrous cycle examined. Both ISG17 and Mx mRNA increased in the stratum compactum ST between Days 11 and 13, and expression extended into the deep GE and stratum spongiosum ST on Days 15 through 17 in pregnant ewes. Interestingly the Mx gene continued to be strongly expressed in LE and superficial GE through Day 17 of pregnancy, whereas ISG17 remained low to undetectable in these cells. Collectively, this study highlights the complexity of the uterine environment by unequivocally illustrating differential temporal and spatial expression of the IFN-responsive genes ISG17 and Mx.

Abstract

We examined the effects of tumour necrosis factor α (TNFα) and interferon γ (IFNγ) on the production of collagen by human infant foreskin fibroblasts. Collagen synthesis was maintained in the presence of IGF-I so that cytokine effects could be examined in the absence of serum. TNFα inhibited IGF-I-maintained collagen production in a dose-dependent manner. Maximal suppression of 50% was attained at a concentration of 7·5 ng/ml. IFNγ also suppressed collagen accumulation in IGF-I-maintained cells, with a maximal inhibition to 55% at 375 U/ml. The rate of collagen formation relative to total protein production for secreted proteins was calculated. This value decreased from 10·3% for IGF-I-cultured cells to 6·2% and 8·4% with the inclusion of TNFα and IFNγ respectively, indicating that inhibition was selective for collagen. TNFα (5 ng/ml) and IFNγ (250 U/ml) together suppressed IGF-I-maintained collagen production to approximately 35%, with a decrease from 10·3% to 2·9% in collagen production relative to total protein. The inclusion of a serum-free period prior to the addition of TNFα to the cultures resulted in a further inhibition to 15% of control. This increase in inhibition was not seen if dexamethasone was present in the serum-free period prior to cytokine addition. These data showed that IGF-Imaintained collagen formation is suppressed by the proinflammatory cytokines TNFα and IFNγ, and that these interactions are influenced by dexamethasone. Proinflammatory cytokines interact in a complex manner with other serum factors to modulate IGF-I-stimulated extracellular matrix production and may have important roles in regulating tissue repair.

Journal of Endocrinology (1995) 147, 167–176

This study aimed at elucidating the effects of interferon (IFN)-alpha on glucose metabolism in patients with chronic hepatitis B and C infections. Twenty-eight biopsy-proven patients with chronic hepatitis B (ten cases) and hepatitis C (18 cases) were given IFN-alpha for a total of 24 weeks. The patients received a 75 g oral glucose tolerance test (OGTT), glucagon stimulation test, tests for type 1 diabetes-related autoantibodies and an insulin suppression test before and after IFN-alpha therapy. Ten of the 28 patients responded to IFN-alpha therapy. Steady-state plasma glucose of the insulin suppression test decreased significantly in responders (13.32+/-1.48 (S.E.M.) vs 11.33+/-1.19 mmol/l, P=0.0501) but not in non-responders (12.29+/-1.24 vs 11.11+/-0.99 mmol/l, P=0.2110) immediately after completion of IFN-alpha treatment. In the oral glucose tolerance test, no significant difference was observed in plasma glucose in either responders (10.17+/-0.23 vs 10.03+/-0.22 mmol/l) or non-responders (10.11+/-0.22 vs 9.97+/-0.21 mmol/l) 3 Months after completion of IFN-alpha treatment. However, significant differences were noted in C-peptide in both responders (2.90+/-0.13 vs 2.20+/-0.09 nmol/l, P=0.0040) and non-responders (2.45+/-0.11 vs 2.22+/-0.08 nmol/l, P=0.0287) before vs after treatment. The changes of C-peptide in an OGTT between responders and non-responders were also significantly different (P=0.0028), with responders reporting a greater reduction in C-peptide. No case developed autoantibodies during the treatment. In patients who were successfully treated with IFN-alpha, insulin sensitivity improved and their plasma glucose stayed at the same level without secreting as much insulin from islet beta-cells.

ABSTRACT

Experiments were performed to determine the mechanism by which recombinant bovine interferon-α_I1 (rbIFN-α) causes an acute reduction in plasma concentrations of progesterone. In experiment 1, administration of a prostaglandin synthesis inhibitor blocked rbIFN-α-induced hyperthermia but did not prevent the decline in plasma concentrations of progesterone. The decline in progesterone concentrations caused by rbIFN-α was, therefore, not a direct consequence of the associated hyperthermia or of pathways mediated through prostaglandin synthesis. It is also unlikely that rbIFN-α acts to increase the clearance of progesterone since injection of rbIFN-α did not decrease plasma concentrations of progesterone in ovariectomized cows given an intravaginal implant of progesterone (experiment 2). In experiment 3, rbIFN-α did not affect basal and LH-induced release of progesterone from cultured luteal slices, indicating that rbIFN-α is unlikely to affect luteal function directly. Injection of rbIFN-α did, however, cause a decrease in plasma concentrations of LH in ovariectomized cows (experiment 4) that coincided temporally with the decrease in progesterone concentrations seen in cows having a functional corpus luteum. The present results strongly suggest that rbIFN-α acts to reduce secretion of progesterone by interfering with pituitary support for luteal synthesis of progesterone. The finding that rbIFN-α can inhibit LH secretion implies that interferon-α molecules should be considered among the cytokines that can regulate hypothalamic or pituitary function.

Journal of Endocrinology (1992) 133, 175–182

Abstract

The sick euthyroid syndrome is a state of altered thyroid hormone metabolism which occurs during illness. The pathogenesis is incompletely understood but recent studies indicate a role of cytokines. It is unknown if cytokines released during illness are directly responsible for the changes in thyroid hormone metabolism. Therefore we studied if previous immunoneutralization of cytokines can prevent endotoxin (lipopolysaccharide LPS), induced sick euthyroid syndrome.

LPS administration resulted in systemic illness, an increase in serum tumor necrosis factor (TNFα) and interleukin (IL)-6 and a decrease in serum triiodothyronine (T₃) and thyroxine (T₄). Immunoneutralization of the effects of cytokines was accomplished by administration of monoclonal antibodies against mouse IL-1 type-1 receptor (IL-1R), TNFα, IL-6 or interferon (IFNα) prior to LPS. The LPS-induced release of cytokines was affected by previous immunoneutralization as compared with control experiments with normal immunoglobulin (IgG): anti-IL-1R did not affect serum TNFα but decreased serum IL-6, anti-TNFα decreased serum TNFα but not IL-6, anti-IL-6 did not affect serum TNFα but hugely increased IL-6 and anti-IFNγ decreased both serum TNFα and IL-6. Specific immunoneutralization of IL-1, TNFα or IFNγ did not prevent the LPS-induced decrease in serum T₃, T₄ and liver 5′-deiodinase mRNA. However, immunoneutralization of IL-6, although not preventing the fall in serum T₃ and T₄, did mitigate the LPS-induced decrease in liver 5′-deiodinase mRNA.

In view of possible non-specific effects of the huge dose of immunoglobulins (1 mg), used only in the immunoneutralization of IL-6, we repeated the experiment with F(ab′)₂ fragments of anti-IL-6 antibodies. Compared with F(ab′)₂ fragments of control IgG, anti-IL-6 F(ab′)₂ did not affect the LPS-induced rise in serum TNFα or the decrease in serum T₃ and T₄ and liver 5′-deiodinase mRNA. Serum IL-6 levels induced by LPS were, however, cleared more rapidly from the circulation when anti-IL-6 F(ab′)₂ fragments rather than intact anti-IL-6 were administered. In conclusion, immunoneutralization of IL-1, TNFα or IFNγ did not prevent the LPS-induced sick euthyroid syndrome in mice; immunoneutralization of IL-6, however, transiently inhibits the LPS-induced decrease of liver 5′-deiodinase mRNA.

Journal of Endocrinology (1997) 153, 115–122

ABSTRACT

A series of experiments was performed to determine whether proteins produced by the sheep conceptus (oCSP) during the time of maternal recognition of pregnancy or bovine recombinant interferon α₁-1 (brIFN) decrease oxytocin receptor concentrations in the endometrium of cyclic or ovariectomized progesterone-treated ewes. In experiment 1, cyclic ewes received intrauterine infusions of serum proteins (oSP), oCSP or brIFN on days 12, 13 and 14 of the oestrous cycle. Ewes then received an oxytocin challenge (1 μg in 0·9% NaCl), and blood samples were taken just before and every 10 min for 1 h after the challenge; these were measured for 13,14-dihydro-15-ketoprostaglandin F_2α (PGFM), the stable metabolite of prostaglandin F_2α. Endometrial oxytocin receptor concentrations were then measured. The oCSP and brIFN treatments suppressed both endometrial oxytocin receptor concentrations and oxytocin-induced increases in PGFM concentrations.

In experiment 2, ewes were ovariectomized and then pretreated with a fluorogestone acetate-releasing intravaginal device for 10 days followed by oestradiol (25 μg i.m. twice daily for 2 days). Ewes were then treated with progesterone (10 mg i.m. twice daily for 12 days). Ewes received intrauterine infusions of oSP, oCSP and brIFN on days 10, 11 and 12 of progesterone treatment. On the day after the last progesterone treatment, ewes were challenged with oxytocin and blood samples collected to measure PGFM. Endometrial oxytocin receptors were also measured. Treatment with oCSP, but not brIFN, suppressed endometrial concentrations of oxytocin receptor, and neither oCSP nor brIFN altered oxytocin-induced increases in PGFM concentrations.

In experiment 3, ewes were ovariectomized and pretreated as in experiment 2 and then received progesterone treatment for 6, 8, 10 or 30 days. On the day after the last progesterone treatment, ewes received an oxytocin challenge and blood samples and endometrium were collected as in experiment 1. Endometrial oxytocin receptors increased sharply between days 8 and 10 and remained raised after 30 days of progesterone treatment. Oxytocin-induced PGFM increased between 8 and 10 days of progesterone treatment, but no response to oxytocin was detected after 30 of progesterone treatment.

In experiment 4, ewes were pretreated as in experiment 2 and then treated for 10 days with progesterone and received intrauterine infusions of oCSP, oSP or brIFN placebo control buffer on days 8, 9 and 10. Ewes received oxytocin and blood samples and endometrium were collected as in experiment 1. As in experiment 2, oCSP treatment suppressed oxytocin receptor concentrations but did not affect oxytocin-induced PGFM release.

In experiment 5, ewes were treated with steroid hormones as in experiment 4 and then received intrauterine infusions of either brIFN or oSP on days 8, 9 and 10 of progesterone treatment. The brIFN treatment suppressed oxytocin receptor concentrations but did not suppress oxytocin-induced increases in plasma PGFM.

We concluded from these experiments that (1) treatment of cyclic ewes with either oCSP or brIFN decreases endometrial oxytocin receptor concentrations and oxytocin-induced increases in PGFM and (2) in progesterone-treated ovariectomized ewes, treatment with oCSP and brIFN suppresses endometrial oxytocin receptor concentrations but does not suppress oxytocin-induced increases in PGFM.

Journal of Endocrinology (1991) 131, 475–482