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Although numerous cytokines, including interleukin (IL)-1, IL-8, and tumor necrosis factor, circulate in critically ill patients at risk for acute respiratory distress syndrome (ARDS), none clearly predict the development of the syndrome. We hypothesized that cytokines, such as IL-1ra, IL-10, and IL-4, which modulate inflammation, might contribute to or reflect the development of acute lung injury. Accordingly, serial levels of IL-1ra and IL-10 were measured in 77 patients who were identifed as being at risk for the development of ARDS. Initial IL-1ra levels were significantly higher (p < 0.0001) in the patients (7.82 [2.29-38.01] ng/ml) than in normal control subjects (0.24 [0.24-0.34] ng/ml) but did not predict the development of ARDS. Initial IL-1ra levels, however, were greater (p = 0.038) in the patients who died (31.95 [3.02-65.06] ng/ml) compared with survivors (6.61 [1.86-29.33] ng/ml). Similarly, IL-10 levels were increased in patients (155 [53.75-318.75] ng/ml) compared with normal control subjects (0 ng/ml) but did not predict the development of ARDS. Like IL-1ra levels, initial IL-10 levels were significantly higher (p = 0.005) in patients who died compared with survivors. IL-4 was not detectable in any of the patient plasma samples measured. Thus, modulators of inflammation are increased in patients at risk for ARDS who die, but do not predict the development of the syndrome.

We show that while interleukin-1 and tumor necrosis factor-alpha antagonists are partially able to inhibit graft-versus-host disease (GVHD) when bone marrow (BM) transplantation is performed in irradiated recipients of minor antigen-disparate donor BM, these same antagonists do not inhibit GVHD when donor BM is fully MHC disparate. This failure in MHC-disparate recipients occurs despite the presence of interleukin-1 and tumor necrosis factor-alpha mRNA in tissues of GVHD mice measured by in situ hybridization and despite escalation of antagonist dosages far beyond those used in previous reports. These findings indicate that a relationship may exist between cytokines that amplify GVHD and target antigens that elicit GVHD. Moreover, when GVHD inhibition was observed in the minor model, it was transient, which suggests that it may be important to target more than one cytokine to effectively inhibit GVHD. These findings suggest that caution is in order in clinical GVHD studies based entirely on anti-proinflammatory cytokine treatment.

Considerable evidence supports the hypothesis that estrogen prevents bone loss by blocking the production of cytokines in bone or bone marrow. However, controversy remains on the role of candidate factors, such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor (TNF). As IL-1 and TNF have many additive and/or synergistic effects in bone, we tested the hypothesis that the simultaneous block of IL-1 and TNF is required to prevent the initial phase of rapid bone loss that follows ovariectomy (ovx). To this aim, rats were ovariectomized and treated for 2 weeks with either IL-1 receptor antagonist (IL-1ra), an inhibitor of IL-1, or TNF-binding protein (TNFbp), an inhibitor of TNF. Ovx increased bone marrow cell secretion of IL-1 and TNF and decreased the bone density of the distal femur, as measured by dual energy x-ray absorptiometry. Ovx-induced bone loss was decreased by both IL-1ra and TNFbp and completely prevented by simultaneous treatment with IL-1ra and TNFbp. Combined treatment with IL-1ra and TNFbp decreased urinary pyridinoline cross-links, a marker of bone resorption that reflects osteoclast number and osteoclast activity, whereas treatment with either inhibitor alone was less effective. Both IL-1ra and TNFbp decreased the number of osteoclasts on the endocortical surfaces and stimulated bone formation, but the two inhibitors had no additive effects on these indexes, suggesting that inhibition of osteoclastogenesis and stimulation of bone formation do not account for the additive bone-sparing effects of IL-1ra and TNFbp. These inhibitors had no effect in sham-operated rats, indicating that they specifically blocked estrogen-dependent events. In conclusion, these data indicate that in the early post-ovx period, IL-1 and TNF play a critical causal role in inducing bone loss and do so by stimulating bone resorption and inhibiting bone formation.

The pathogenic role of interleukin-1 (IL-1) in the progression of established rat crescentic glomerulonephritis was investigated by administration of the interleukin-1 receptor antagonist (IL-1ra). Passive accelerated antiglomerular basement membrane (GBM) disease was induced in three groups of six rats. One group was killed on day 7 with no treatment. The other groups received a constant infusion of IL-1ra or saline from day 7 until being killed on day 21. All animals developed moderate glomerular injury, a significant loss of renal function and marked histological damage including crescent formation by day 7. Saline treated animals showed a significant deterioration in these parameters over days 7 to 21. In contrast, animals treated with the IL-1ra over this period showed stabilization of glomerular injury (protein-uria; P < 0.001) and a recovery of normal renal function (creatinine clearance; P < 0.05). Histologically, IL-1ra treatment suppressed glomerular cell proliferation (PCNA expression; P < 0.001) and significantly inhibited crescent formation (P < 0.005), glomerular sclerosis (P < 0.005), tubular atrophy (P < 0.05) and interstitial fibrosis (P < 0.05). A key finding was that IL-1ra treatment not only stopped renal leukocyte accumulation over days 7 to 21 (P < 0.01), but that treatment also suppressed immune activation of the infiltrate (P < 0.01). In conclusion, this study provides direct evidence that IL-1 plays a key role in the progressive/chronic phase of renal injury in experimental crescentic glomerulonephritis and indicates that IL-1ra treatment may be of therapeutic benefit in human rapidly progressive crescentic glomerulonephritis.

Interleukin-1 receptor antagonist (IL-1ra) competes with IL-1 for binding of the IL-1 receptor, but does not elicit a cellular immune response. This study was designed to evaluate the effectiveness of IL-1ra in the immune and inflammatory responses to rat heart allografts. Experimental design was as follows: Group I HTx was syngeneic, BN to BN. The remaining groups were DA (RT 1a) to BN (RT 1n) allogeneic HTx. Group II was transplanted without immunosuppression. Group III received a low-dose IL-1ra regimen via osmotic pump into the peritoneum. Group IV recipients were similarly treated with a higher dose IL-1ra regimen. Group V rats received subtherapeutic cyclosporine (CsA) therapy while Group VI was treated with both CsA and low-dose IL-1ra. Group I rats survived indefinitely. Group II rats rejected their grafts at 5.33 +/- 1.37 days. Group III grafts survived for 7.16 +/- 0.48 days, and Group IV grafts for 8.16 +/- 0.75 days, both significantly longer than in Group II (P < 0.01). Group V animals treated with low-dose CsA had graft survival of 7.7 +/- 1.6 days, but combined therapy with CsA and IL-1ra in Group VI yielded significantly prolonged graft survival of 17.2 +/- 1.3 days (P < 0.0001). Histologic examination in treated recipients revealed delayed appearance of mononuclear cell infiltration. IL-1ra-treated recipients all demonstrated significantly reduced numbers of graft-infiltrating leukocytes; all phenotype subsets were equally affected. This study demonstrates the effectiveness of IL-1ra, in combination with low-dose CsA, in reducing the inflammatory response and rejection in the transplant setting.

To investigate the contribution of IL-1, IL-6, and TNF to the increased osteoclastogenesis induced by estrogen deficiency, ovariectomized (ovx) mice were treated with either IL-1 receptor antagonist (IL-1ra), a competitive inhibitor of IL-1, TNF binding protein (TNFbp), an inhibitor of TNF, or the anti-IL-6 antibody (Ab) 20F3 for the first 2 wk after surgery. ovx increased the bone marrow cells secretion of IL-1 and TNF, but not IL-6, and the formation of TRAP-positive osteoclast-like multinucleated cells (MNCs) in bone marrow cultures treated with 1,25(OH)2D3. The increase in MNC formation induced by ovx was prevented by in vivo treatment with either 17 beta estradiol, IL-1ra, TNFbp, or anti-IL-6 Ab. However, the percent change in MNC formation induced by the anti-IL-6 Ab was similar in ovx and sham-operated animals, whereas IL-1ra and TNFbp were effective only in ovx mice. MNC formation was also decreased by in vitro treatment of bone marrow cultures with IL-1ra and TNFbp, but not with anti-IL-6 Ab. Ovx also increased bone resorption in vivo and in vitro, as assessed by the urinary excretion of pyridinoline cross links and the formation of resorption pits, respectively. IL-1ra, TNFbp and estrogen decreased bone resorption in vivo and in vitro whereas the anti-IL-6 Ab inhibited bone resorption in vitro but not in vivo. In conclusion, these data indicate that IL-1 and TNF play a direct role in mediating the effects of ovx on osteoclastogenesis and bone resorption. The data also suggest that IL-6 is not essential for increasing bone resorption in the early postovariectomy period.

The recent finding that treatment with the interleukin-1 (IL-1) inhibitor, interleukin-1 receptor antagonist (IL-1ra) decreases bone loss and bone resorption in ovariectomized rats, strongly suggested that IL-1 mediates, at least in part, the effects of estrogen deficiency on bone resorption. Although in vitro studies have shown that IL-1 activates mature osteoclasts and stimulates osteoclastogenesis, the two main mechanisms by which estrogen deficiency stimulates bone resorption, it is still unclear whether IL-1 mediates both effects of estrogen deficiency in vivo. To investigate this matter, we have examined the changes in bone mineral density (BMD) which occur in ovariectomized rats after completion of 1 month of estrogen or IL-1ra treatment begun at the time of ovariectomy. Ovariectomy caused a marked decreased in BMD which was blocked by 17 beta estradiol and decreased by IL-1ra. Cessation of estrogen therapy was followed by a rapid induction of bone loss, indicating that estrogen blocks the activation and utilization of mature osteoclasts without depleting the bone microenvironment of osteoclast precursors and mature, inactive osteoclasts. In contrast, ovariectomized rats treated with IL-1ra maintained a stable bone density for the first 4 weeks after completion of the treatment. In these rats, bone loss resumed not earlier than 6 weeks after discontinuation of the IL-1ra treatment. Estrogen deficiency was necessary to unveil the bone-sparing effect of IL-1ra because in a control experiment in which rats were treated with IL-1ra for the 4 weeks before ovariectomy, BMD began to decrease immediately after ovariectomy.(ABSTRACT TRUNCATED AT 250 WORDS)

Systemic administration of recombinant human interleukin-1 receptor antagonist (IL-1Ra) caused a rapid and sustained elevation of plasma IL-1Ra levels and decreased the leak of intravascularly injected 125I-labeled albumin into lungs of rats given human recombinant interleukin-1 intratracheally. IL-1Ra treatment decreased leak when given 0.5 h before, 1.25 h after, or 2.5 h after IL-1 administration. Similarly, IL-1Ra treatment decreased lavage leukocytes and neutrophils when given 0.5 h before, 1.25 h after, or 2.5 h after IL-1 administration. Pretreatment with IL-1Ra also decreased lung myeloperoxidase activity and breath H2O2 concentration increases in rats given IL-1 intratracheally. Our results suggest that IL-1Ra treatment may decrease acute lung injury and neutrophil influx even when given after an IL-1 inciting insult.

Interleukin-1 (IL-1), a cytokine produced by bone marrow cells and bone cells, has been implicated in the pathogenesis of postmenopausal osteoporosis because of its potent stimulatory effects on bone resorption in vitro and in vivo. To investigate whether IL-1 plays a direct causal role in post ovariectomy bone loss, 6-mo-old ovariectomized rats were treated with subcutaneous infusions of IL-1 receptor antagonist (IL-1ra), a specific competitor of IL-1, for 4 wk, beginning either at the time of surgery or 4 wk after ovariectomy. The bone density of the distal femur was measured non invasively by dual-energy X-ray absorptiometry. Bone turnover was assessed by bone histomorphometry and by measuring serum osteocalcin, a marker of bone formation, and the urinary excretion of pyridinoline cross-links, a marker of bone resorption. Ovariectomy caused a rapid increase in bone turnover and a marked decrease in bone density which were blocked by treatment with 17 beta estradiol. Ovariectomy also increased the production of IL-1 from cultured bone marrow cells. Ovariectomy induced-bone loss was significantly decreased by IL-1ra treatment started at the time of ovariectomy and completely blocked by IL-1ra treatment begun 4 wk after ovariectomy. In both studies IL-1ra also decreased bone resorption in a manner similar to estrogen, while it had no effect on bone formation. In contrast, treatment with IL-1ra had no effect on the bone density and the bone turnover of sham-operated rats, indicating that IL-1ra specifically blocked estrogen-dependent bone loss. In conclusion, these data indicate that IL-1, or mediators induced by IL-1, play an important causal role in the mechanism by which ovariectomy induces bone loss in rats, especially following the immediate post ovariectomy period.

Interleukin-1 is a proinflammatory cytokine produced in glomerulonephritis. Blocking the action of interleukin-1 by the administration of the interleukin-1 receptor antagonist (IL-1ra) has been shown to prevent renal function impairment, reduce glomerular injury, inhibit leukocyte infiltration, and suppress tubulointerstitial damage in experimental antiglomerular basement membrane disease. A key mechanism in the entry of leukocytes into the kidney is the interaction between the interleukin-1 inducible intercellular adhesion molecule-1 (ICAM-1; CD54) and lymphocyte function-associated antigen-1 (CD11a/CD18). Therefore, this study investigated whether the inhibition of this mechanism was the means by which IL-1ra suppressed leukocyte infiltration in rat accelerated antiglomerular basement membrane glomerulonephritis. Disease was induced in two groups of six rats; animals were treated by constant sc infusion of recombinant human IL-1ra or saline from the initiation of disease until being euthanized 14 days later. In saline-treated animals, there was marked up-regulation of ICAM-1 in the glomerulus and interstitium, In which was associated with leukocyte infiltration. In particular, focal accumulation of CD11a+ and CD18+ cells was apparent in areas of tubulointerstitial damage exhibiting intense ICAM-1 expression. IL-1ra treatment partially reduced glomerular ICAM-1 expression and leukocyte infiltration. However, IL-1ra treatment resulted in a dramatic inhibition of interstitial ICAM-1 expression, interstitial leukocyte infiltration, and tubulointerstitial damage. In conclusion, this study has shown that interleukin-1 is a major inducer of ICAM-1 expression within the renal tubulo-interstitium--a process associated with focal leukocyte infiltration and tubulointerstitial damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Circulating concentrations of tumor necrosis factor-alpha (TNF), interleukin (IL)-1 beta, IL-6, IL-1 receptor antagonist (IL-1ra), and soluble TNF receptors p55 (sTNFr-55) and p75 (sTNFr-75) and ex vivo production of TNF, IL-1, IL-6, and IL-1ra using a whole blood culture system were measured during the acute and convalescent stages of meningococcal infection. Circulating TNF and IL-1 were below detection level, whereas IL-6 and IL-1ra, sTNFr-55, and sTNFr-75 were increased at admission. The ex vivo production of proinflammatory cytokines TNF, IL-1, and IL-6 was suppressed at admission and restored gradually during recovery. On the contrary, the production of the antiinflammatory IL-1ra was increased at admission. The elevated concentrations of both IL-1ra and sTNFr early in the course of infection suggest a regulatory role for these antiinflammatory compounds. The observed down-regulation of the ex vivo production of TNF, IL-1, and IL-6 and up-regulation of the production of IL-1ra in the acute stage may indicate a protective regulation mechanism.

The contribution of IL-1 to leukocyte infiltration in anti-glomerular basement membrane (GBM) antibody (Ab) glomerulonephritis (GN) was examined by the administration of a specific IL-1 receptor antagonist (IL-1ra). Lewis rats received anti-GBM Ab or normal rabbit serum and were treated with either 0.9% saline or 6 mg IL-1ra over a 24-h time period. Plasma IL-1ra concentration was 2,659 +/- 51 ng/ml 4 h after anti-GBM Ab and IL-1ra administration. PMN and monocyte/macrophage infiltration declined 39% (9.8 +/- 1.9 to 6.0 +/- 1.5 PMN/glomerulus, P < 0.001) and 29% (4.9 +/- 0.8 to 3.5 +/- 0.8 ED-1 cells/glomerulus, P = 0.002) with IL-1ra treatment at 4 h, respectively. Similarly, the number of glomerular cells staining for lymphocyte function-associated molecule-1 beta (CD18) declined 39% from 16.7 +/- 1.9 to 10.7 +/- 1.6 cells/glomerulus at 4 h (P = 0.0001). This was associated with a decrease in glomerular intracellular adhesion molecule-1 expression. The mean glomerular intracellular adhesion molecule-1 score in anti-GBM Ab GN rats treated with IL-1ra was less than that of rats administered anti-GBM Ab and 0.9% saline at 4 (2.0 +/- 0.2 vs 2.5 +/- 0.2, P < 0.05) and 24 (2.5 +/- 0.1 vs 3.1 +/- 0.2, P = 0.0001) h. These immunopathologic changes correlated with a 50% reduction in proteinuria from 147 +/- 34 to 75 +/- 25 mg/d (P < 0.002). Treatment with IL-1ra did not affect the steady state mRNA expression of either IL-1 beta or TNF alpha. An increase in the IL-1ra dose to 30 mg given within the initial 4 h provided no additional benefit. The decline in PMN and monocyte/macrophage infiltration of the glomerulus at 4 h was similar to that found in the initial study. Furthermore, the protective benefit of IL-1ra was abrogated by doubling the dose of the anti-GBM Ab GN, despite administering high dose IL-1ra (30 mg). In these studies, detectable IL-1ra was found in the serum of untreated anti-GBM Ab GN controls. These data suggest a positive yet limited role for IL-1ra in the therapeutic intervention of anti-GBM Ab GN.

Interleukin-1 (IL-1), a potent stimulant of bone resorption, has been implicated in the pathogenesis of postmenopausal osteoporosis, because monocyte IL-1 bioactivity increases after the menopause and is decreased by estrogen and progesterone (EP) replacement. As IL-1 bioactivity reflect the production of both IL-1 and the IL-1 inhibitor, IL-1 receptor antagonist (IL-1ra), EP treatment could decrease IL-1 bioactivity by regulating the secretion of either IL-1 or IL-1ra. We now report that EP treatment in vivo decreased the secretion into the medium of cultured monocytes of IL-1ra and IL-1 beta as well as the IL-1 beta/IL-1ra ratio. We also found that in normal women the production of IL-1ra was within premenopausal levels in the first 7 yr after the menopause and increased linearly thereafter. In these women, monocyte IL-1 beta, IL-1 beta/IL-1ra ratio, and IL-1 bioactivity were all increased in the first 7 yr after the menopause and within the premenopausal range thereafter. In osteoporotic women, IL-1 beta, IL-1 beta/IL-1ra ratio, and IL-1 bioactivity increased after the menopause and returned to premenopausal levels after 15 yr from the menopause. In these women monocyte IL-1ra secretion was above the premenopausal range at all times after the menopause, but did not change with the passage of time since menopause. We conclude that hormone replacement decreases the in vitro secretion of both IL-1ra and IL-1 beta. The data also suggest that in normal women a progressive increase in the secretion of IL-1ra contributes to restore a normal IL-1/IL-1ra ratio after the menopause, a phenomenon which, in turn, may play a role in limiting postmenopausal bone loss.

Background: This study was designed to determine the role of interleukin-1 (IL-1) in hemorrhagic shock death., Methods: Pentobarbital anesthetized C3H/HeN mice (n = 59) were prepared with a femoral arterial catheter and were randomized to treatment with an IL-1 receptor antagonist (IL-1ra, 10 mg/kg, n = 29) or an equal volume of phosphate-buffered saline solution (vehicle, n = 30) by subcutaneous bolus injection at 15 minutes before hemorrhage and again at 120 minutes. Continuous posthemorrhage delivery of IL-1ra or vehicle was performed in each group (1.5 mg IL-1ra in 30 microliters/day) through a subcutaneous osmotic pump. Rapid hemorrhage of 4 ml/100 gm weight was followed by normal saline resuscitation of 12 ml/100 gm 60 minutes later., Results: Survival analysis by Wilcoxon rank sum analysis revealed a significantly improved 5-day survival in IL-1ra-treated mice (n = 15, 20%) as compared with vehicle-treated mice (n = 14, 6%, p < 0.001). To determine a possible mechanism of this survival advantage, the remaining mice in each treatment group were killed at 30 minutes to obtain blood and tissue samples from the heart, liver, and kidney for measurement of adenosine-5'-triphosphate (ATP). No difference in hematocrit, circulating neutrophils, or levels of glucose, lactate, or tumor necrosis factor was identified between groups to explain the improved outcome. IL-1ra prevented hemorrhage-induced ATP depletion observed in vital organs of vehicle-treated mice., Conclusions: The data implicate IL-1 in shock-induced ATP depletion and suggest IL-1ra may improve hemorrhagic shock survival by preventing ATP depletion in vital organs.

The role of interleukin-1 (IL-1) in the pathogenesis of experimental crescentic glomerulonephritis was investigated. Administration of the interleukin-1 receptor antagonist (IL-1ra) was used to block the action of IL-1 during disease development. Two groups of six rats were primed with rabbit IgG, followed five days later by injection of rabbit anti-GBM serum (day 0). Animals were treated with a constant infusion of recombinant human IL-1ra (plasma level approximately 100 to 200 ng/ml) or saline (untreated) from day -1 until being killed on day 14. Untreated animals exhibited severe proteinuria and development renal dysfunction shown by increased serum urea and serum creatinine and reduced creatinine clearance. In contrast, IL-1ra treated animals had significantly reduced proteinuria (IL-1ra vs. untreated, P < 0.05) and maintained normal renal function (IL-1ra vs. untreated, P < 0.05). Histologically, IL-1ra treatment markedly reduced glomerular hypercellularity, glomerular necrosis and crescent formation and almost completely abrogated tubular atrophy and fibrosis. IL-1ra treatment suppressed glomerular macrophage accumulation by 57% (P < 0.01), while macrophage accumulation in the interstitium was completely abrogated and immune activation of the interstitial T cell infiltrate was prevented. This study demonstrates that IL-1 plays a key role in the pathogenesis of anti-GBM glomerulonephritis, and blocking its effects may provide a novel therapeutic approach to the treatment of human progressive glomerulonephritis.

Interleukin 1 (IL-1) is a cytokine released during immune activation that mediates the host's response to infection and inflammation. Peripheral and central injections of IL-1 induce fever and sickness behavior, including decreased food motivation and reduced interest in social activities. To determine the receptor mechanisms responsible for these effects, rats were injected with IL-1 receptor antagonist (IL-1ra), an endogenous cytokine that acts as a pure antagonist of IL-1 receptors. IL-1ra blocked the increased body temperature and oxygen consumption induced by injection of recombinant human IL-1 only when both cytokines were administered i.p. In contrast, i.p. or intracerebroventricular administration of IL-1ra blocked the depressive effect of IL-1 beta on food-motivated behavior and social exploration when this cytokine was administered by the same route as the antagonist. In addition, intracerebroventricular IL-1ra blocked the reduction in social exploration produced by i.p. IL-1 beta but had only partial antagonist effects on the decrease in food-motivated behavior induced by i.p. IL-1 beta. In each case, the dose of IL-1ra was 100- to 1000-fold in excess of the biologically active dose of IL-1. These results suggest that the receptor mechanisms that mediate the behavioral and pyrogenic effects of IL-1 are heterogeneous.

An inhibitor of tumor necrosis factor (TNF) has been isolated from the human histiocytic lymphoma cell line U-937 that is capable of inhibiting both TNF-alpha and TNF-beta. Protein sequencing has verified that it is distinct from a previously described TNF inhibitor that is a soluble fragment of a TNF receptor molecule (TNFrI). The cDNA sequence of this second TNF inhibitor clone suggests that it is also a soluble fragment of a TNF receptor. Expression of this cDNA sequence in COS-7 cells verified that it encodes a receptor for TNF-alpha (TNFrII) that can give rise to a soluble inhibitor of TNF-alpha, presumably through proteolytic cleavage. The extracellular domain of TNFrII has significant homology with that of TNFrI and these two receptors share a striking conservation of cysteine residue alignment with the extracellular domain of the nerve growth factor receptor. These three receptor molecules are therefore members of a family of polypeptide hormone receptors.

We present pharmacological and genetic evidence that regulation of different genes by glucocorticoid hormones in the rat hepatoma cell line, HTC, occurs in a coordinate manner. We have analyzed the responses of four different glucocorticoid-inducible proteins, tyrosine aminotransferase [L-tyrosine:2-oxoglutarate aminotransferase (EC 2.6.1.5)], glutamine synthetase [L-glutamate:ammonia ligase (EC 6.3.1.2)], a secreted glycoprotein Belt I, and the mouse mammary tumor virus (MMTV)-encoded protein (gp52) in these cells. The concentration of dexamethasone necessary for half-maximal induction of each of these proteins is 10-20 nM, the same concentration necessary to half-saturate glucocorticoid receptors. Furthermore, glucocorticoids of varying potency elicit parallel inductions of these markers. MSN5.3, a glucocorticoid receptor-defective cell line selected for its inability to induce gp52, is also unable to induce the other three cellular gene products. In contrast, another class of variants incapable of gp52 induction retains inducibility of the other three markers. We show here by "superinfection" with MMTV that these cells harbor a defect in the original integrated provirus itself and not in the cellular induction machinery. The results presented here suggest that the induction of glucocorticoid-responsive genes in these cells is mediated by a single glucocorticoid induction pathway.

alpha 1-acid glycoprotein (alpha 1-AGP), or orosomucoid, is shown to be inducible by glucocorticoids in HTC rat hepatoma cells. Immunoprecipitation of [35S]methionine pulse-labeled proteins from these cells reveals secreted proteins of Mr = 35,000-48,000 (alpha 1-AGP) and Mr greater than 180,000, both of which are greatly enhanced by glucocorticoid treatment. The amount of alpha 1-AGP-specific mRNA in HTC cells is greatly increased (at least 100-fold) in response to glucocorticoids. The new steady-state level of RNA is approached with a t 1/2 of about 8 hr and the RNA consists of a single species of approximately 850 bases. The response is specific for glucocorticoids since: (i) the EC50 for dexamethasone is 30 nM; (ii) the glucocorticoid antagonist, progesterone, inhibits the induction by dexamethasone; and (iii) a glucocorticoid receptor-deficient cell line is incapable of alpha 1-AGP mRNA induction. This is a secondary hormonal response since inhibition of protein synthesis blocks the induction of alpha 1-AGP mRNA by dexamethasone whereas the induction of mouse mammary tumor virus (MMTV) RNA is unaffected.

Using Chinese hamster ovary (CHO) cells transfected with a plasmid carrying the human beta-interferon gene, we find that inhibitors of protein synthesis, in the absence of any other inducer, stimulate the production of interferon RNA; this effect is maintained in cells in which the plasmid sequences have been amplified 25- to 50-fold. Nuclear transcription assays show that a major effect of cycloheximide is to increase the rate of transcription of the interferon gene. This contradicts the generally accepted explanation that inhibitors of protein synthesis augment interferon production by stabilizing interferon mRNA. In addition, we have studied the effects of double stranded RNA [poly(rI) X poly(rC)] on the induction of interferon RNA in the presence and absence of cycloheximide. Our results indicate that poly(rI) X poly(rC) by itself causes a transient increase in interferon RNA; however, in the presence of cycloheximide this effect is prolonged. We do not, however, find an increase in transcription of the interferon gene(s) as an early response to poly(rI) X poly(rC). Finally, we have found that cells treated with cycloheximide or infected with Newcastle disease virus induce large amounts of a secreted 11-kDa protein. This cellular protein is not inducible by poly(rI) X poly(rC). We propose that both interferon and this 11-kDa protein belong to a family of proteins in which production is regulated in a coordinate fashion during viral inhibition of cellular protein synthesis.

We have studied the glucocorticoid-mediated accumulation of alpha 1-acid glycoprotein (AGP) in mRNA in HTC rat hepatoma cells. In contrast to the well-characterized primary response of mouse mammary tumor virus, in vitro transcription assays in isolated nuclei show that the rate of transcription of the AGP gene is high even in the absence of hormone. Despite the constitutive transcription of the AGP gene, no detectable AGP RNA can be found in either the cytoplasm or the nuclei of untreated cells. Previous experiments have shown that the glucocorticoid induction of AGP RNA requires ongoing protein synthesis. In conjunction with the present study, our data suggest that glucocorticoids stimulate accumulation of AGP RNA by inducing an RNA processing factor that allows production of stable transcripts.

The complete nucleotide sequence of the rat alpha 1-acid glycoprotein gene has been determined from an isolated lambda recombinant bacteriophage. Southern blot analysis and DNA sequencing indicate that there is only one gene per genome; it contains six exons and is located within a 3,200-base-pair fragment starting from a TATA box and extending to the polyadenylation signal AATAAA. Transcription starts 37 base pairs upstream from the beginning of the translation codon ATG. The TATA box (TATAAA) lies 26 base pairs upstream from this site. The gene contains several potential glucocorticoid receptor-binding sites, both inside and outside the structural gene.

Ciliary neurotrophic factor (CNTF) is one of a small number of proteins with neurotrophic activities distinct from nerve growth factor (NGF). CNTF has now been purified and cloned and the primary structure of CNTF from rabbit sciatic nerve has been determined. Biologically active CNTF has been transiently expressed from a rabbit complementary DNA clone. CNTF is a neural effector without significant sequence homologies to any previously reported protein.

The genome of human hepatitis B virus (HBV) contains an enhancer element within a 360-base-pair transcribed region located between sequences encoding the virus surface and core antigens. Beyond the usual properties associated with enhancers (i.e., activity 5' or 3' of a heterologous promoter and relative orientation independence), DNA sequences encompassing this enhancer exhibited unexpected characteristics. Using gene expression assays in both stably and transiently transfected cells, we found that the HBV enhancer element, when located in a transcribed region of chimeric genes, dramatically increased expression levels of genes controlled by the simian virus 40 promoter/enhancer. This synergism was not observed, however, when the HBV enhancer was located outside of the transcribed region. When these transcribed sequences were reversed in orientation, expression levels decreased significantly. These data suggest that RNA stability and transcriptional activity may be affected by sequences associated with this DNA region. In contrast to the findings of others, we found that the HBV enhancer activated transcription in a relatively cell-type-independent manner when the enhancer was located either 5' of the promoter or in the 3' untranslated region of gene constructs. The implications of these and other properties of the HBV enhancer region on viral gene expression and its life cycle are discussed.

Transforming growth factor alpha (TGF-alpha) is a transformation-responsive mitogenic polypeptide that is expressed in the brain, epithelial cells, and activated macrophages. We isolated and characterized the TGF-alpha promoter and localized the 5' end of the TGF-alpha transcript to a unique position. Surprisingly, no apparent TATA box was present in the promoter sequence, suggesting that transcription from mammalian genes can initiate at unique and specific positions from promoters lacking this sequence motif.