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Adoptive immunotherapy using receptor-modified T lymphocytes has shown promise in preclinical studies for the treatment of infectious and malignant diseases. These modified T cells express chimeric receptors that link ligand recognition and signal transduction domains in a single gene product. Typically, a single chain Fv fragment is genetically attached to the cytoplasmic domain of the T-cell receptor (TCR) zeta chain. Modulating the signaling characteristics of chimeric receptors will be important for their application to human immunotherapy. It was hypothesized that linking coreceptor and costimulatory signaling motifs together with the zeta signaling domain will enhance receptor function. The present study compares signaling characteristics of 9 single-chain receptors consisting of the H-2K(b) extracellular and transmembrane domains and various combinations of T cell signal transduction domains. Signal transduction regions studied include the TCR zeta chain, the CD4 coreceptor, the lck protein tyrosine kinase, and the CD28 costimulatory receptor. Biochemical characteristics of the receptors, analyzed using calcium flux, receptor, and ZAP-70 phosphorylation, and lck association may be predicted from the known functions of receptor constituents. The combination of zeta together with coreceptor and costimulatory function in a single receptor maximizes chimeric receptor sensitivity and potency. Combining zeta with either the costimulatory or coreceptor function independently also enhances receptor function, though to a lesser extent. It is therefore possible to link TCR, coreceptor, and costimulatory activities in a single functional entity using modular domains. Such receptors demonstrate distinct signaling properties and should prove useful in the development of chimeric receptors for therapeutic purposes.

We have used the selective farnesylation inhibitor HR12 [cysteine-N(methyl)valine-N(cyclohexyl) glycine-methionine-O-methyl-ester] to study the role of oncogenic Ras in cytoskeletal reorganization in Ha-ras(V12)-transformed Rat1 cells (Rat1/ras). Application of HR12 resulted in complete restoration of the cytoskeleton and associated cell adhesions disrupted by oncogenic Ras. This included an increase in the number and size of focal adhesions, accompanied by massive stress fiber formation and enhanced tyrosine phosphorylation. Furthermore, HR12 induced assembly of adherens junctions and dramatically elevated the level of the junctional components, cadherin and beta-catenin. HR12 was unable to restore the nontransformed phenotype in cells expressing farnesylation-independent, myristylated Ras. Examination of the main Ras-regulated signaling pathways revealed that HR12 induced a dose- and time-dependent decline in Erk1&2 activation (t(1/2) approximately 6 h), which correlated with the accumulation of nonfarnesylated oncogenic-Ras. Inhibition of the Mek/Erk pathway in Rat1/ras cells, using the Mek inhibitor, PD98059, resulted in complete cytoskeletal recovery, indistinguishable from that induced by HR12. Moreover, a constitutively active Mek mimicked the effect of ras transformation in Rat1 cells, and prevented HR12-induced cytoskeletal effects in Rat1/ras cells. No such effects were observed after treatment of Rat1/ras cells with the phosphatidylinositol 3-kinase inhibitor LY294002. These findings establish the Mek/Erk pathway as the dominant pathway involved in conferring the cytoskeletal and junctional manifestations of the Ras-induced transformed phenotype.

The present study was performed to identify a potent and sequence-specific antisense oligonucleotide (ASO), to inhibit Hdm2 expression in human cancer cell lines and to study the downstream consequences. Ten chimeric 2'-O-methoxyethyl (MoE)-modified hemimers were synthesized that targeted various regions from the 5'- to the 3'-end of Hdm2 mRNA. The IC50 of the most potent ASO, NCH-4401, was subsequently determined and compared to the IC50 of a 2'-MoE-modified ASO, with a complete phosphorothioate backbone (NCH-4668), and to a 3 bp mismatched ASO (NCH4529). NCH4401 inhibited Hdm2 expression in SJSA-1 cells with an IC50 of 120 nm, whereas NCH-4668 was less potent with an IC50 of 180 nm. The mismatched control ASO was completely inactive, indicating a sequence-dependent mechanism of action of NCH-4401. NCH4401 was subsequently used to study the consequences of inhibiting Hdm2 expression in human osteosarcoma cells. NCH-4401 completely inhibited Hdm2 protein expression in SJSA-1 cells at a concentration of 300 nm, already 4 h after start of ASO treatment. At an ASO concentration of 300 nM, p53 protein was induced 12.5-fold and p21 was induced 8-fold over background levels, 24 h after start of ASO treatment. The dramatic induction of p53 in SJSA-1 cells prompted us to investigate whether the accumulation of p53 in these cells was followed by induction of apoptosis. However, no signs for apoptosis were detected in SJSA-1 cells, following induction of wild-type p53 using the Yopro method and the induction of caspase-3 activity. SJSA-1 cells were subsequently treated with NCH-4401 at different concentrations in combination with two well-known DNA-damaging agents, i.e. carboplatin and mitomycin C. Apoptosis induction following treatment of cells with DNA-damaging agents and NCH4401 was determined in parallel by measuring caspase-3 activation and uptake of the DNA dye Yopro. Carboplatin and mitomycin C together only slightly induced apoptosis in SJSA-1 cells to a factor of approximately 2-fold, as measured by the induction of caspase-3 activity. The downregulation of Hdm2 expression by NCH4401 did not induce apoptosis on its own and did not potentiate the mitomycin C/carboplatin-induced programmed cell death.

The progress made in understanding the molecular basis of mammalian cell transformation has led to the unifying concept of growth regulation and its disorders in cancer cells. Today it is well recognized that many products of "cancer genes" encode for proteins that regulate normal mitogenesis and apoptosis. Taken together, this indicates that the carcinogenic process may be viewed as a progressive disorder of signal transduction (1-6). In fact, many of the genes that are mutated or lost in cancer cells, including both the oncogenes and tumor suppressors, encode proteins that are crucial regulators for intra- as well as intercellular signal transduction (1-6). This conceptual framework has provided a basis for the development of novel anticancer strategies and therapeutic modalities with the aim to inhibit cancer growth either by blocking mitogenic signal transduction or to specifically induce apoptosis of cancer cells. Although these various approaches have not been validated clinically, these strategies are likely to identify compounds with less side effects compared to standard chemotherapeutic agents.

Three distinct waves of Leydig cell development are found in the pig testes, which occur during fetal, perinatal, and prepubertal periods. Proliferation of Leydig cells is primarily regulated by luteinizing hormone (LH); however, effects of LH on proliferation of immature rat Leydig cells are mediated by specific growth factors and cytokines such as transforming growth factor-alpha (TGFalpha), insulin-like growth factor-1 (IGF-1), interleukin-1beta (IL-1beta), steroidogenesis-inducing protein (SIP), and TGFbeta. The objective of the present study was to identify growth factors that could possibly be involved in the proliferation of Leydig cells in the neonatal pig testis. Leydig cells were isolated from 3- to 5-d-old pig testes, cultured for 48 hr in serum-free media, washed, and treated with hCG and/or IGF-1, epidermal growth factor (EGF), IL-1beta, SIP, and TGFbeta for 18 hr. Tritiated thymidine incorporation into DNA was assessed over a subsequent 4-hr period. Incorporation of [3H]-thymidine was stimulated by hCG treatment with a 2.3-fold increase over control cultures. SIP also induced a significant increase (P < 0.0001) in the incorporation of [3H]thymidine into Leydig cell DNA. Similarly, EGF and IGF-1 also increased DNA synthesis in neonatal porcine Leydig cells, whereas IL-1beta had no effect. TGFbeta had very little, if any, effect on DNA synthesis when added alone, but inhibited the stimulatory effects of other mitogens (SIP, hCG, EGF/TGFalpha, and IGF-1). Our results indicate that these growth factors may play a role in the LH/hCG-dependent proliferation of Leydig cells during the perinatal period of development.

The TCR complex signals through a set of 10 intracytoplasmic motifs, termed immunoreceptor tyrosine-based activation motifs (ITAMs), contained within the gamma-, delta-, epsilon-, and zeta-chains. The need for this number of ITAMs is uncertain. Limited and contradictory studies have examined the ability of subsets of the TCR's ITAMs to signal into postthymic primary T lymphocytes. To study signaling by a restricted set of ITAMs, we expressed in transgenic mice a chimeric construct containing the IAs class II MHC extracellular and transmembrane domains linked to the cytoplasmic domain of the TCR zeta-chain. Tyrosine phosphorylation and receptor cocapping studies indicate that this chimeric receptor signals T cells independently of the remainder of the TCR. We show that CD4+ and CD8+ primary T cells, as well as naive and memory T cells, are fully responsive to stimulation through the IAs-zeta receptor. Further, IAs-zeta stimulation can induce primary T cell differentiation into CTL, Th1, and Th2 type cells. These results show that the zeta-chain ITAMs, in the absence of the gamma, delta, and epsilon ITAMs, are sufficient for the activation and functional maturation of primary T lymphocytes. It also supports the isolated use of the zeta-chain ITAMs in the development of surrogate TCRs for therapeutic purposes.

A 20-mer phosphorothioate oligodeoxynucleotide (ODN) directed against human protein kinase C-alpha (CGP 64128A = ISIS 3521) was analyzed for its antitumor activity either alone or in combination therapy. Combination studies with CGP 64128A and standard chemotherapeutic agents (cisplatin, mitomycin-C, vinblastine, estracyt and adriamycin) were performed in nude mice that had been transplanted s.c. with a variety of human tumors (breast, prostate, large cell lung and small cell lung carcinomas, and melanomas). Additive antitumor effects with CGP 64128A and the cytotoxins were found for half of the combinations studied. The combination of CGP 64128A with vinblastine or cisplatin showed superadditive antitumor activities against MCF-7 human breast carcinomas and PC3 prostate carcinomas with complete responses. CGP 64128A in combination with adriamycin resulted in superadditive antitumor effects against BT20 human breast carcinomas with complete tumor responses, and in combination with mitomycin-C superadditive antitumor effects with cures were observed against NCI-H460 human large cell carcinomas. The antitumor activity of CGP 64128A appeared to be due to a sequence-dependent mechanism of action as two 20-mer control ODNs were completely inactive as single agents against A549 and NCI-H69 human lung carcinomas. The antitumor activity of cisplatin against NCI-H69 human small cell lung carcinomas was slightly inhibited by one of the control ODNs, indicating that the superadditive antitumor activities of CGP 64128A in combination with cisplatin are the result of a sequence-dependent mechanism of action.

A 20-mer phosphorothioate oligodeoxynucleotide (ODN) directed against human C-raf kinase (CGP 69846A or ISIS 5132) was analyzed for its antitumor activity either alone or in combination therapy. Combination studies with CGP 69846A and standard chemotherapeutic agents (cisplatin, mitomycin C, tamoxifen, or Adriamycin) were performed in nude mice that had been transplanted s.c. with a variety of human tumors (breast, prostate, colon, small cell lung, large cell lung, and squamous lung carcinomas). For the majority of the combinations studied, additive antitumor effects with CGP 69846A and the cytotoxins were found. The combination of CGP 69846A with cisplatin or mitomycin C showed superadditive antitumor activities against NCI-H69 small cell lung carcinomas with complete tumor responses. CGP 69846A, in combination with cisplatin, showed superadditive antitumor effects against PC3 human prostate carcinomas with tumor cures, and in combination with mitomycin C, superadditive antitumor effects of CGP 69846A with tumor cures against NCI-H460 large cell lung carcinoma were found. These effects appeared to be sequence-specific because a mismatched control ODN was completely without effect as a single agent against NCI-H69 human small cell lung cancers. The combination of the mismatched control ODN with mitomycin C or cisplatin did not influence the antitumor activity of the cytotoxins against NCI-H69 human small cell lung cancers, indicating that the superadditive antitumor effects observed for CGP 69846A in combination with cisplatin or mitomycin C are the result of a sequence-specific mechanism of action in NCI-H69 human small cell lung cancers.

To determine the mechanism of action responsible for the in vivo antitumor activity of a phosphorothioate antisense inhibitor targeted against human C-raf kinase (ISIS 5132, also known as CGP69846A), a series of mismatched phosphorothioate analogs of ISIS 5132 or CGP69846A were synthesized and characterized with respect to hybridization affinity, inhibitory effects on C-raf gene expression in vitro, and antitumor activity in vivo. Incorporation of a single mismatch into the sequence of ISIS 5132 or CGP69846A resulted in reduced hybridization affinity toward C-raf RNA sequences and reduced inhibitory activity against C-raf expression in vitro and tumor growth in vivo. Moreover, incorporation of additional mismatches resulted in further loss of in vitro and in vivo activity in a manner that correlated well with a hybridization-based (i.e., antisense) mechanism of action. These results provide important experimental evidence supporting an antisense mechanism of action underlying the in vivo antitumor activity displayed by ISIS 5132 or CGP69846A.

A 20-mer phosphorothioate oligodeoxynucleotide (ISIS 3521) designed to hybridize sequences in the 3'-untranslated region of human protein kinase C-alpha (PKC-alpha) mRNA has been shown to inhibit the expression of PKC-alpha in multiple human cell lines. In human bladder carcinoma (T-24) cells, inhibition of PKC-alpha was both concentration dependent and oligonucleotide sequence specific. ISIS 3521 had a IC50 of 50-100 nM for PKC-alpha mRNA reduction and was without effect on the expression of other members of the PKC family of genes (PKC-eta and zeta). Toxicity studies in mice revealed that the oligodeoxynucleotide was well tolerated at repeat doses of 100 mg/kg i.v. for up to 14 days, with no acute toxicity apparent. The oligodeoxynucleotide was found to also inhibit the growth of three different human tumor cell lines, the T-24 bladder, human lung carcinoma (A549), and Colo 205 colon carcinoma grown in nude mice. The inhibition was dose dependent with ID50 values for the growth inhibition between 0.06 and 0.6 mg/kg daily when given i.v., depending on the cell line examined. Three control phosphorothioate oligodeoxynucleotides not targeting human PKC-alpha were without effect on the growth of the tumors at doses as high as 6 mg/kg. Recovery of ISIS 3521 from tumor tissue and resolution by capillary gel electrophoresis revealed that 24 It after the final dose of oligodeoxynucleotide, intact, full-length 20-mer material was present as well as some apparent exonuclease degradation products (e.g., n-1 and n-2 mers). These studies demonstrate the in vivo antitumor effects of an antisense oligodeoxynucleotide targeting PKC-alpha and suggest that this compound may be of value as a chemotherapeutic agent in the treatment of human cancers.

Substantial evidence exists supporting a direct role for raf kinases in the development and maintenance of certain human malignancies. Here we test the potential of phosphorothioate antisense oligodeoxynucleotides targeted against human C-raf-1 kinase to specifically inhibit C-raf-1 kinase gene expression and tumor progression in cell culture and in vivo, using human tumor xenograft mouse models. Treatment of human tumor cells with appropriate phosphorothioate antisense oligodeoxynucleotides led to specific inhibition of C-raf kinase gene expression in cell culture and in vivo at well-tolerated doses. Moreover, oligodeoxynucleotide treatment resulted in potent antiproliferative effects in cell culture and potent antitumor effects in vivo against a variety of tumor types that were highly consistent with an antisense mechanism of action for these compounds. These studies strongly suggest that antisense inhibitors targeted against C-raf-1 kinase may be of considerable value as antineoplastic agents that display activity against a wide spectrum of tumor types at well-tolerated doses.

A 20 mer oligodeoxynucleotide designed to hybridize to specific 3'-untranslated sequences in the type I receptor IL-1r mRNA was identified which inhibited the expression of both IL-1r mRNA and protein in human A549 lung carcinoma cells and human dermal fibroblasts. The oligodeoxynucleotide exhibited an IC50 value of approximately 100 nM in both cell lines and reduced IL-1r mRNA expression for up to 48 h. Multiple scrambled control oligonucleotides were without effect on IL-1r mRNA expression. Treatment of A549 cells with this oligodeoxynucleotide (but not scrambled controls) inhibited the IL-1 stimulated expression of the cell adhesion molecule ICAM-1 but was without effect on the TNF-alpha induction of this molecule. This study demonstrates that phosphorothioate oligodeoxynucleotides can selectively inhibit IL-1r expression leading to a reduction in IL-1 dependent gene expression.

A major process through which the immune system becomes tolerant to self proteins involves the deletion of self reactive cells in the thymus. However, T cells reactive to peripheral tissue-specific proteins can escape this deletion and become tolerized in the periphery by a variety of mechanisms. We report here, contrary to expectation, that the pancreas-specific protein, elastase I, is also expressed at a low level in the thymus, and that this thymic expression contributes to tolerance induction. To study the mechanism of this tolerance induction, we utilized a double transgenic mouse model. In these mice the expression of a model protein, SV40 T antigen, is directed by the elastase I promoter and hence parallels elastase I expression in the pancreas and thymus. These mice were crossed with mice transgenic for a TCR specific for T antigen, so the majority of thymocytes and T cells in these mice express the transgene. In double transgenic mice we find that thymic expression of T antigen results in anergic thymocytes which also show a reduction of Th1 activity with no decrease in Th2 activity. These functional characteristics persist in peripheral T cells, but there is also a depletion in the number of T antigen reactive T cells in lymph nodes. Chimeras were constructed which directly demonstrated that the thymus is the site of tolerance induction and that the tolerizing element is thymic epithelium. We propose that the loss of Th1 activity as a consequence of the thymic epithelium being encountered by tissue-specific proteins results in the functional tolerization of CTL in vivo, despite the fact that CTL are fully functional in vitro. In this way autoimmune destruction is contained. Thymic expression of peripheral proteins may therefore be an additional way in which tolerance to peripheral proteins can be achieved.

Objective: CGP 47969A is a novel and potent inhibitor of cytokine biosynthesis in human and murine monocytic cells. The effect of CGP 47969A on collagen induced arthritis (CIA) in DBA/1 mice was investigated and compared to that of prednisolone., Methods: CIA was induced by intradermal injection of type II collagen in CFA at Day 0. CGP 47969A was applied orally, 5 times/week, starting at Day 15 after immunization. Arthritic signs were recorded 3 times/week for clinical scoring and radiographic analysis was performed at the end of the experiment at Day 60. Serum amyloid P (SAP) and anticollagen antibody titers were determined by ELISA at Day 60., Results: CGP 47969A dose dependently reduced the incidence of arthritis from 93% in the positive control group to 60, 40, 30 and 10% at doses of 1, 5, 25 and 60 mg/kg/day, respectively. At a dose of 120 mg/kg, CGP 47969A totally prevented the occurrence of arthritis (ED50 between 1 and 5 mg/kg). Prednisolone at 3 and 30 mg/kg reduced the arthritis incidence to 70 and 30%, respectively. CGP 47969A dose dependently inhibited the joint destruction, as measured by radiographic scoring and its potency was comparable to that of prednisolone. The elevated serum levels of the positive acute phase protein SAP in arthritic animals were completely normalized by CGP 47969A at a dose of 60 mg/kg, however, neither CGP 47969A nor prednisolone influenced the plasma levels of anticollagen antibodies (IgG)., Conclusion: Our results demonstrate that CGP 47969A is highly effective in CIA with a potency comparable to that of prednisolone. These promising results justify the expectation that this novel antiarthritic compound now under development might also be effective in the treatment of rheumatoid arthritis in man.

CGP 47969A is a novel piperazine derivative that inhibits the synthesis of inflammatory cytokines, such as interleukin-1 alpha (IL-1), IL-1 beta and tumor necrosis factor alpha (TNF), in human monocytes stimulated with lipopolysaccharide (LPS), zymosan or IL-1 itself. IC50 values are in the range of 0.3-5 mumol/l. CGP 47969A does not inhibit total protein or RNA synthesis indicating selectivity for cytokine inhibition. CGP 47969A exerts its inhibitory effect at a post-transcriptional level, most probably by reducing translational efficiency of IL-beta mRNA, as steady-state levels of IL-1 beta mRNA are not inhibited while the primary translation product, the 31 kD IL-1 beta precursor molecule, is dose-dependently inhibited by CGP 47969A. The compound is devoid of cyclooxygenase and phospholipase A2 inhibitory activity but efficiently inhibits the generation of PGE2 and LTC4 in zymosan-stimulated mouse macrophages with an IC50 of 1.2 and 0.6 mumol/l, respectively. Antagonism of IL-1 and/or TNF is thought to have a beneficial effect on the course of inflammatory diseases. CGP 47969A may therefore represent a mechanistically new approach to the treatment of such diseases.

The qualities of a peripheral Ag that determine whether T cells will be tolerant of or responsive to it are poorly understood. To approach this problem, we studied the T cell response in a line of transgenic mice selectively expressing an oncoprotein in the islets of Langerhans. The SV40 large tumor Ag (SV40-T) is directed to islet beta-cells in Rip1-Tag3 (RT3) mice by a hybrid insulin promoter-SV40-T construct. Ag is first detected on these cells between 10 and 12 wk after birth. RT3 mice were bred with mice expressing a transgenic rearranged TCR recognizing SV40-T in the context of the class I MHC molecule, H-2Kk. T cell response in the resultant RT3/TCR-double transgenic mice was then analyzed. T cells are fully responsive to SV40-T in RT3/TCR-transgenic mice, and T cells infiltrate the islets of both RT3 and RT3/TCR-transgenic mice. This work demonstrates that T cells may remain responsive to self-Ag expressed outside the thymus, and that this responsiveness may result in autoimmunity. The developmentally delayed expression or the oncogenic nature of SV40-T in the RT3-transgenic mice may be important in determining this T cell response.

Interleukin-1 (IL-1) has been implicated in the tissue destruction of rheumatoid arthritis, a disease that is widely treated with glucocorticoids. In this study we investigated the effect of the T cell product interferon-gamma (IFN-gamma) on the glucocorticoid-mediated and on the IL-4-mediated inhibition of IL-1 beta mRNA and IL-1 beta protein synthesis in highly purified human monocytes. Both dexamethasone and IL-4 dose-dependently inhibited IL-1 beta mRNA and IL-1 beta protein synthesis after stimulation with LPS (300 ng/ml); maximal inhibition of 80-90% was achieved. IFN-gamma (1-100 U/ml) did not influence IL-1 beta mRNA and IL-1 beta protein levels in unstimulated cells, but potentiated the LPS-induced synthesis of IL-1 beta mRNA and IL-1 beta protein. After a preincubation time of 1 h, 100 U/ml of IFN-gamma increased the LPS-induced IL-1 beta production by about 20-40%. When human monocytes were preincubated for 1 h with IFN-gamma (100 U/ml) prior to the addition of dexamethasone (10(-6) M) and prior to the stimulation with LPS, the dexamethasone-mediated inhibition of IL-1 beta mRNA and IL-1 beta protein synthesis was totally neutralized by IFN-gamma. In addition, IFN-gamma totally overcame the negative effect of IL-4 (100 pM) on IL-1 beta protein synthesis. A preincubation period of at least 1 h with IFN-gamma was necessary for the neutralization of the dexamethasone effect. If IFN-gamma was given at the same time or after dexamethasone, only a weak effect was found.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-1 (IL-1) has been implicated in the development and progression of a variety of acute and chronic inflammatory diseases. Due to its pro-inflammatory and tissue-degrading activities, IL-1 is regarded as a major mediator of chronic inflammatory joint diseases, including rheumatoid arthritis in man, adjuvant arthritis in rats and collagen-induced arthritis in mice. However, conclusive experimental evidence for the crucial role of IL-1 in the development of joint destruction has not been presented as yet. In the present study, we investigated the effect of a neutralizing monoclonal mouse antibody against mouse IL-1 beta (IgG1 isotype) on the development and progression of collagen-induced arthritis in DBA/1 mice. The antibody was injected intraperitoneally 3 times a week, either from day 3 or from day 21 after primary immunization, to day 60. In the positive control group an arthritis incidence of 80% was observed after 60 days. The injection of a control antibody of the same isotype did not influence the incidence of arthritis, whereas injection of anti-IL-1 beta from day 21 reduced the arthritis incidence to about 30%. Injection of anti-IL-1 beta starting at day 3 totally prevented both the development of arthritis and the associated increase of the acute phase protein serum amyloid P (SAP). Anti-collagen antibody titers, which increased significantly after immunization, were not influenced by the injection of anti-IL-1 beta antibodies, in spite of the suppressive effect on arthritis development.(ABSTRACT TRUNCATED AT 250 WORDS)

We have used our newly described mouse tissue chamber model [1], to investigate the process of IL-1 production in more detail. The inflammatory reaction in the tissue surrounding the implanted chambers was investigated histologically and by using the polymerase chain reaction (PCR). The inflammatory response included influx of leucocytes into the granuloma surrounding the tissue chamber, expression of IL-1 beta on macrophages present in the inflamed tissue and an increase in the mRNA coding for IL-1 beta and IL-6 proteins in the granuloma. The effects of three anti-inflammatory or immunosuppressive drugs, prednisolone, indomethacin and cyclosporin A, on IL-1 beta and PGE2 production in zymosan and Bordetella-pertussis-vaccine (BPV)-challenged tissue chambers were also examined. Oral treatment with prednisolone and cyclosporin A of zymosan-challenged animals showed a dose-dependent reduction of IL-1 beta concentrations, but no effect of indomethacin. Both prednisolone and indomethacin dose-dependently reduced PGE2 concentrations to control levels, while cyclosporin A was effective only at the highest dose tested (100 mg/kg/day p.o.). In drug-treated BPV-challenged animals, prednisolone and cyclosporin A also showed a dose-dependent reduction of IL-1 beta, while indomethacin was again ineffective. Prednisolone and indomethacin also dose-dependently reduced the PGE2 concentrations to control levels, whereas cyclosporin A was effective only at the highest dose tested (100 mg/kg/day p.o.). This model will be useful for investigating the mechanisms controlling the production of IL-1 beta from the mRNA level to the secretion of mature biologically active protein [1], and in the search for new drugs which could selectively interfere with this process.

A simple and reliable animal model to quantify interleukin-1 (IL-1) production at a site of inflammation has been developed and characterised. This model involves the subcutaneous implantation of sterile Teflon chambers (30 mm x 10 mm diameter) into the backs of mice. After 14 days, a straw coloured transudate fluid was present in the lumen of the implanted chamber which was withdrawn for the determination of baseline measurements of various inflammatory parameters. A localised chronic inflammatory response was then induced in the chambers by injection of 1% zymosan or Bordetella pertussis vaccine (BPV) (in presensitised animals). The local inflammatory reaction in the chamber, over a 30 day time course, was characterised by leucocyte infiltration, and marked increases in protein, prostaglandin E2, IL-1 and IL-6 concentrations in the chamber fluid. A rapid increase in plasma concentrations of the acute-phase reactant serum amyloid P (SAP) also occurred. This model allows repeated samples to be obtained from the same animal for the assessment of inflammatory parameters and may be useful for investigating the mechanisms controlling the production of IL-1 during the inflammatory response in vivo.

Using specific cDNA probes, we have investigated drug-induced changes in hepatic mRNA levels of the major acute-phase proteins (APP) fibrinogen, alpha 2-macroglobulin (alpha 2-MG), albumin and alpha 1-acid glycoprotein (alpha 1-AGP) in male Lewis rats with adjuvant arthritis. Test compounds were given orally from day 0 to 20 and hepatic mRNA analysis was performed at day 21. Prednisolone (1, 3, 10 mg/kg), Cyclosporine A (1, 3, 10 mg/kg) and cyclophosphamide (3 mg/kg) dose-dependently normalized hepatic mRNA levels of all four APP. Equipotent anti-inflammatory doses of indomethacin (0.3, 1 mg/kg) significantly downregulated alpha 2-MG mRNA levels but were much less effective in influencing albumin and alpha 1-AGP mRNA levels and even slightly increased hepatic fibrinogen mRNA levels. These results suggest that cytokine over-production, which is thought to be responsible for the acute-phase response in rats with adjuvant arthritis, can be effectively downregulated by immunosuppressive drugs, but is distinctly less affected by the cyclooxygenase inhibitor indomethacin.

Using specific cDNA probes, we have investigated changes in hepatic mRNA concentrations of the major acute phase proteins fibrinogen, alpha 2-macroglobulin (alpha 2-MG), albumin and alpha 1-acid glycoprotein (alpha 1-AGP) during developing adjuvant arthritis in Lewis rats. Continuously increasing levels in the mRNA of the positive reactants beta-fibrinogen, alpha 2-MG and alpha 1-AGP were found during developing disease with peak levels from day 15 to 21, whereas mRNA concentrations of the negative reactant albumin decreased, reaching their lowest levels on day 11 to 15. As early as 4 days after arthritis induction, the hepatic mRNA levels of beta-fibrinogen, alpha 1-AGP and albumin were distinctly different from control values. The most dramatic changes in the hepatic mRNA levels and plasma concentrations of acute phase reactants were seen between days 11 and 21. These results indicate that overproduction of the major inflammatory cytokines IL-1, TNF-alpha and IL-6, which are now felt to be largely responsible for the acute phase response in the rat, is an early event during adjuvant arthritis and that the highest amounts are produced during the inflammatory phase of the disease. mRNA changes in the acute phase proteins alpha 1-AGP and albumin, which are mainly regulated by IL-1/TNF alpha, were more pronounced than those of alpha 2-MG and beta-fibrinogen, which are predominantly controlled by IL-6.(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment of rat mesangial cells with interleukin-1 beta (IL-1 beta) and forskolin induced, in a synergistic fashion, the expression of group II phospholipase A2 (PLA2) mRNA, with subsequent increased synthesis and secretion of PLA2. In contrast, interleukin-6 did not increase PLA2 mRNA levels of PLA2 activity. Transforming growth factor (TGF) beta 1, TGF beta 2 and TGF beta 3 equipotently attenuated the IL-1 beta- and forskolin-induced elevation of PLA2 mRNA, as well as PLA2 synthesis and secretion. The glucocorticoid dexamethasone only partially suppressed the IL-1 beta- and forskolin-induced elevation of PLA2 mRNA, but totally inhibited PLA2 synthesis and secretion.

The effect of recombinant human C5a (rhC5a) on the synthesis of interleukin 1 beta (IL-1 beta) was investigated in human monocytes, isolated by leukapheresis and countercurrent elutriation. rhC5a induced IL-1 beta mRNA synthesis in a dose- and time-dependent manner. Maximal induction was achieved at 3 h with rhC5a concentrations of 200 to 500 ng/ml. The maximal rhC5a-stimulated mRNA induction was about 75% of that observed using LPS as the stimulus (300 ng/ml). The inducing activity of rhC5a could be neutralized by preincubation with a polyclonal anti-C5a antibody. On the other hand rhC5a in optimal concentrations only weakly stimulated IL-1 beta protein synthesis, as measured by a two-site directed enzyme-linked immunoassay. When compared on Northern blots, a slightly reduced mobility of C5a-stimulated IL-1 beta mRNA was observed relative to LPS-stimulated RNA. To exclude the possibility that structural defects in the IL-1 beta mRNA are responsible for the weak translational efficiency after C5a stimulation a primer extension experiment was performed. No difference in the length of the extended fragment was detected between LPS and C5a-stimulated RNA, suggesting that the 5'-regions of the RNAs are identical. When LPS- or C5a-stimulated RNA was used to program IL-1 beta synthesis in an in vitro translation system from rabbit reticulocytes, no difference in translational efficiency was observed. Our results indicate that in human monocytes two signals for IL-1 beta gene expression are necessary, a signal for transcriptional activation and another signal to induce translation of the mRNA.

Treatment of rat mesangial cells with interleukin 1 beta (IL-1 beta) and forskolin greatly enhanced the expression of group II phospholipase A2 (PLA2) mRNA, with subsequent increased synthesis and secretion of PLA2, as detected by PLA2 activity measurements and immunoprecipitation of culture media of [35S]methionine-labelled mesangial cells. PDGF-BB dose-dependently suppressed the IL-1 beta- and forskolin-induced elevation of PLA2 mRNA, as well as PLA2 synthesis and secretion. In contrast, PDGF-AA had no inhibitory effect. The tyrosine kinase inhibitor genistein dose-dependently antagonized the inhibitory effect of PDGF-BB on IL-1 beta-stimulated PLA2 secretion, thus suggesting that tyrosine phosphorylation may be required for PDGF-BB inhibition of PLA2 gene expression in mesangial cells.

The monokines interleukin-1 beta and alpha (IL-1) play a central role in the connective tissue destruction of many chronic inflammatory diseases. A high capacity screening assay for the detection of inhibitors of IL-1 biosynthesis has been established. Normal human monocytes were obtained by leukapheresis and elutriation. IL-1 beta and alpha biosynthesis was stimulated with LPS, and cell-associated and secreted IL-1 beta and IL-1 alpha were measured by specific immunoassays (ELISA). The mean total IL-1 beta (cell-associated and secreted) production in 18 different donors was 11 ng/10(6) cells (range 1.2-28.8). Secreted IL-1 beta represented 31 to 86% of the total IL-1 beta. More IL-1 alpha than IL-1 beta was produced but, unlike IL-1 beta, IL-1 alpha was poorly secreted. The steroids prednisolone and dexamethasone, gold (sodium aurothiomalate) and chloroquine were potent inhibitors of the IL-1 production. Mean IC50 values of 180 nM (range 2.5 nM-1 microns), 10 microM (range 6-20 microM) and of 75 microM were found for prednisolone, gold and chloroquine, respectively. Above 5 microM, the non-steroidal anti-inflammatory compounds indomethacin and BW755C increased IL-1 beta biosynthesis. Nordihydroguaiaretic acid inhibited the level of the secreted form of IL-1 beta, but tended to increase the cell-associated level. D-Penicillamine (up to 6 mM), cyclosporin A (up to 1 microM) and methotrexate (up to 12 microM) inhibited neither cell-associated nor secreted IL-1 beta levels. This high capacity assay, which is insensitive to classical NSAIDs, may serve in the detection and characterization of new classes of anti-inflammatory compounds.

Rat alpha 1-inhibitor 3 clones were isolated by immunological screening of a lambda gt11 cDNA library prepared from rat liver poly(A)-rich RNA. The recombinant cDNA clones were identified by the absence of their immunoprecipitable products following hybrid-arrested in vitro translation. The size of the cognate poly(A)-rich RNA was estimated to be roughly 5000 residues. Approximately 16 h after induction of inflammation the amount of alpha 1-inhibitor 3 poly(A)-rich RNA decreases as shown by dot-blot hybridization and Northern analyses. The response of this negative acute-phase plasma protein to inflammation may therefore be considered to be at the pretranslational level. The characterized DNA constitutes an open reading frame of 225 amino acids followed by a canonical eucaryotic polyadenylation signal and a poly(A) tail. Sequence microheterogeneity, particularly in the 3'-flanking region was observed. An amino acid homology of 70% for alpha 1-inhibitor 3 with human and rodent alpha 2-macroglobulin emphasizes the evolutionary relationship of the macroglobulins.

The three monokines interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), and interleukin-6 (IL-6) modulate acute phase plasma protein synthesis in adult human hepatocytes. Only IL-6 stimulates the synthesis of the full spectrum of acute phase proteins as seen in inflammatory states in humans, i.e. synthesis and secretion of C-reactive protein, serum amyloid A, fibrinogen, alpha 1-antitrypsin, alpha 1-antichymotrypsin and haptoglobin are increased while albumin, transferrin and fibronectin are decreased. IL-1 beta as well as TNF alpha, although having a moderate effect on the positive acute phase proteins and inhibiting the synthesis of fibrinogen, albumin and transferrin, fail to induce serum amyloid A and C-reactive protein. These data suggest that IL-6 plays the key role in the regulation of acute phase protein synthesis in human hepatocytes.

Secretory products of cultured human blood monocytes contain a hepatocyte-stimulating factor which is able to induce the acute-phase proteins alpha 2-macroglobulin and fibrinogen in rat liver cells. Total RNA was isolated from unstimulated and lipopolysaccharide-stimulated human monocytes and translated in a reticulocyte lysate. The capability of the cell-free synthesized proteins to induce the acute-phase proteins alpha 2-macroglobulin and fibrinogen was assayed in rat hepatocyte primary cultures and in the rat hepatoma cell line Fao. The products translated from the mRNA of lipopolysaccharide-stimulated human monocytes induced mRNAs for alpha 2-macroglobulin and fibrinogen and therefore contain hepatocyte-stimulating factor. The translation products of unstimulated monocytes had no effect. A cDNA containing the coding sequence for interleukin-6 (B-cell stimulatory factor 2, interferon-beta 2/26-kDa protein, interleukin HP1) derived from human T-cells cloned into the transcription vector pGEM4 was transcribed in vitro. Translation of the isolated RNA in a reticulocyte lysate led to the synthesis of a protein of about 25 kDa. This cell-free synthesized interleukin-6 exhibited hepatocyte-stimulating activity measured by the induction of beta-fibrinogen mRNA in Fao cells. Using an antibody against interleukin-6, two proteins of 22 kDa and 23 kDa were immunoprecipitated from the culture medium of lipopolysaccharide-stimulated human monocytes. These two proteins were not synthesized by unstimulated monocytes. When total RNA from unstimulated human monocytes and lipopolysaccharide-stimulated human monocytes and lymphocytes was subjected to Northern analysis and hybridized with the interleukin-6 cDNA, a strong hybridization signal corresponding to an RNA of about 1300 bases was detected only in the RNA from lipopolysaccharide-stimulated human monocytes, indicating that human monocytes express the interleukin-6 gene after stimulation. The data presented in this paper strongly suggest that hepatocyte-stimulating factor from human monocytes and interleukin-6 from T-cells are identical.

Conditioned medium from human monocytes contains a partially characterized hepatocyte-stimulating factor that simultaneously elevates the mRNA levels of the acute-phase protein beta-fibrinogen and decreases albumin mRNA in rat hepatoma cells. We demonstrate that recombinant human B-cell stimulatory factor 2, which is identical to interferon-beta 2/26 kDa protein and interleukin-HP1, exhibits the same activity as hepatocyte-stimulating factor. Furthermore, a specific antibody against B-cell stimulatory factor 2 was able to inhibit hepatocyte-stimulating factor in conditioned medium from human monocytes. Our data show that hepatocyte-stimulating factor and B-cell stimulatory factor 2 are functionally and immunologically related proteins.

The plasma half-life of recombinant human interleukin-6 (rhIL-6) was determined in rats by measuring the disappearance of the biological activity as well as of the radioactivity of 125I-rhIL-6 from the circulation. The kinetics of clearance were biphasic. It consisted of a rapid initial disappearance corresponding to a half-life of 3 min, and of a second slow one corresponding to a half-life of about 55 min. By cellulose-acetate electrophoresis it was shown that rhIL-6 binds to a plasma protein resulting in a complex migrating in the beta-gamma region; 20 min after intravenous injection, about 80% of the 125I-rhIL-6 that had disappeared from the circulation was found in the liver. 125I-rhIL-6 was exclusively localized on the surface of parenchymal cells suggesting the existence of an interleukin-6 receptor on the hepatocytes.

Recombinant human interleukin 6 (rhIL 6) was injected i.p. into male Wistar rats to investigate its role as a mediator of the acute-phase response. Hepatic mRNA levels of beta-fibrinogen, alpha 2-macroglobulin, cysteine proteinase inhibitor, alpha 1-acid glycoprotein and albumin were measured at different times after the administration of rhIL 6. Maximal increases of mRNA concentrations were observed already 4 h after the injection of rhIL 6 leading to 4.8-, 19.7-, 10- and 16-fold stimulations in mRNA levels of beta-fibrinogen, alpha 2-macroglobulin, cysteine proteinase inhibitor or alpha 1-acid glycoprotein, respectively. The rhIL 6-induced stimulation of acute-phase protein mRNA was much more rapid than the acute-phase induction after turpentine, where maximal mRNA levels were found between 16 and 24 h. For all acute-phase proteins studied, the stimulation of mRNA synthesis was found to be dependent on the dose of rhIL 6 injected. In the case of alpha 2-macroglobulin mRNA a sex-specific induction by rhIL 6 was found. Only male rats showed an acute-phase response, whereas in female rats an acute-phase reaction of alpha 2-macroglobulin mRNA was not inducible by IL 6. The increases in mRNA levels of the acute-phase proteins studied were followed by corresponding changes of the proteins in the serum determined by rocket immunoelectrophoresis. It is concluded that IL 6 represents a potent mediator of the acute-phase response in the rat.

The rat hepatoma cell line Fao was used to study the role of three inflammatory mediators on the mRNA regulation of several acute-phase proteins. In the presence of 10(-6) M dexamethasone beta-fibrinogen mRNA levels increased 6-fold after addition of recombinant human IL 6 (rhIL 6). rhIL 1 beta or recombinant human tumor necrosis factor alpha (rhTNF alpha) had essentially no effect on beta-fibrinogen mRNA induction but led to a 20-fold increase in alpha 1-acid glycoprotein mRNA in the presence of dexamethasone. On the other hand, rhIL 6 was a much weaker stimulator of alpha 1-acid glycoprotein mRNA synthesis. All three mediators reduced albumin mRNA concentrations to about 30% of controls. Whereas the induction of beta-fibrinogen mRNA was potentiated by dexamethasone, the synthetic glucocorticoid analog was an absolute requirement for the stimulation of alpha 1-acid glycoprotein mRNA. The mRNA levels of the negative acute-phase protein albumin were induced 5-fold by dexamethasone alone. The beta-fibrinogen mRNA induction started immediately after addition of rhIL 6 and reached a maximum between 12 and 18 h. In contrast, the time-course for alpha 1-acid glycoprotein mRNA synthesis showed a lag phase of 8 h followed by an increase up to 20 h after rhIL 1 beta. rhTNF alpha led to an even more delayed increase in alpha 1-acid glycoprotein mRNA. Whereas in the case of beta-fibrinogen mRNA induction no synergistic effect was observed between various concentrations of the three mediators, the combination of rhIL 6/rhIL 1 beta as well as rhIL 6/rhTNF alpha or rhIL 1 beta/rhTNF alpha regulated synergistically alpha 1-acid glycoprotein and albumin mRNA. It is concluded that discrete acute-phase proteins are regulated differently by the inflammatory mediators IL 6, IL 1 beta and TNF alpha, indicating that the acute-phase response is more complex than previously assumed. The Fao cell line used in this study turned out to be an ideal model for acute-phase protein regulation, suitable for the discrimination between the inflammatory mediators IL 6 and IL 1/TNF alpha.

The regulation of the three major acute-phase proteins alpha 2-macroglobulin, cysteine proteinase inhibitor and alpha 1-antitrypsin by recombinant human interleukin-1 beta, recombinant human interleukin-6 and recombinant human tumor necrosis factor alpha was studied in rat hepatocyte primary cultures. Synthesis and secretion of the acute-phase proteins was measured after labeling with [35S]methionine and immunoprecipitation. Incubation of hepatocytes with interleukin-6 led to dose-dependent and time-dependent changes in the synthesis of the three major acute-phase proteins and albumin, similar to those occurring in vivo during experimental inflammation. alpha 2-Macroglobulin and cysteine proteinase inhibitor synthesis was induced 54-fold and 8-fold, respectively, 24 h after the addition of 100 units/ml interleukin-6. At the same time synthesis of the negative acute-phase protein albumin was reduced to 30% of controls. Half-maximal effects were achieved with 4 units interleukin-6/ml. Interleukin-1 beta had only a partial effect on the regulation of the four patients studied: only a twofold stimulation of alpha 2-macroglobulin and a 60% reduction of albumin synthesis were observed. Tumor necrosis factor alpha did not alter the synthesis of acute-phase proteins. The stimulation of alpha 2-macroglobulin and cysteine proteinase inhibitor synthesis by interleukin-6 was inhibited by interleukin-1 beta in a dose-dependent manner. In pulse-chase experiments the effect of interleukin-1 beta, interleukin-6 and tumor necrosis factor alpha on the secretion of acute-phase proteins was examined. Interleukin-6 markedly accelerated the secretion of total proteins and alpha 2-macroglobulin, whereas the secretion of cysteine proteinase inhibitor, alpha 1-antitrypsin and albumin was not affected. The inhibition of N-glycosylation by tunicamycin abolished the effect of interleukin-6 on the secretion of alpha 2-macroglobulin, indicating a possible role of interleukin-6 on N-glycosylation.

The biosynthesis and secretion of alpha 1-antitrypsin was studied in rat hepatocyte primary cultures. After labeling with [35S]methionine an alpha 1-antitrypsin with an apparent molecular weight of 49000 estimated by sodium dodecyl sulfate/polyacrylamide slab gel electrophoresis was immunoprecipitated from the cell homogenate. This intracellular form of alpha 1-antitrypsin could be deglycosylated by endoglycosidase H treatment indicating that its oligosaccharide chains were of the high-mannose type. Pulse-chase experiments showed that about 30 min after its synthesis the transformation of the 49000-Mr alpha 1-antitrypsin to a protein with an apparent molecular weight of 54000 began. Only this 54000-Mr protein was secreted by the hepatocytes. The 54000-Mr alpha 1-antitrypsin was not sensitive to endoglycosidase H, but sensitive to neuraminidase, and it incorporated [3H]galactose and [3H]fucose indicating that its oligosaccharide chains were of the complex type. In the presence of tunicamycin, which blocks the formation of N-asparagine-linked oligosaccharide chains, an unglycosylated alpha 1-antitrypsin with an apparent molecular weight of 41000 was found in the cells as well as in the medium. However, tunicamycin decreased the secretion of alpha 1-antitrypsin by 60-70%, whereas the secretion of albumin remained unaffected. In the presence of colchicine the secretion of both alpha 1-antitrypsin and albumin was impaired. The results demonstrate the importance of glycosylation for the secretion of alpha 1-antitrypsin.

The biosynthesis of rat alpha 1-inhibitor3, a negative acute-phase reactant specifically found in rodents, was studied in vitro in a cell-free translation system from rabbit reticulocytes, in rat hepatocyte primary cultures and in vivo by immunocytochemistry using normal and turpentine-injected rats. By sucrose-gradient centrifugation and subsequent translation of the fractionated RNA in vitro it was found that the mRNA coding for alpha 1-inhibitor3 exhibited a size of about 28S. For the alpha 1-inhibitor3 translated in vitro an apparent Mr of 155,000 was determined. A continuous decrease in the level of alpha 1-inhibitor3 in serum during experimental inflammation induced by turpentine injection was demonstrated by means of quantitative 'rocket' immunoelectrophoresis. This result agrees with the observation by immunocytochemistry of a drastic decrease in alpha 1-inhibitor3 levels in hepatocytes 24 h after turpentine injection. At that time alpha 1-inhibitor3 is mainly located in the Golgi apparatus, whereas it is also present in the membranes of the rough and smooth endoplasmic reticulum when normal liver is used. All hepatocytes, but no other hepatic cells, contain alpha 1-inhibitor3. When hepatocyte primary cultures were labelled with [35S]methionine and alpha 1-inhibitor3 was immunoprecipitated from the hepatocyte medium and the supernatant of homogenized cells, two different forms of alpha 1-inhibitor3 were found. The intracellular form of alpha 1-inhibitor3, with an apparent Mr of 173,000, is characterized by oligosaccharide side chains of the high-mannose type. The form of alpha 1-inhibitor3 in the medium exhibited an Mr of 186,000 and carried carbohydrate side chains of the complex type. After labelling hepatocytes with radioactive sugars, [3H]mannose was found in both forms of alpha 1-inhibitor3, whereas [3H]fucose and [3H]galactose were incorporated only into the form found in the medium. In the presence of tunicamycin an unglycosylated alpha 1-inhibitor3 with an apparent Mr of 154,000 was found in cells and in the medium. In a pulse-chase experiment it was shown that inhibition of glycosylation by tunicamycin resulted in a marked delay of secretion of alpha 1-inhibitor3. Thus the oligosaccharide side chains of alpha 1-inhibitor3 play an important role during its transport into the medium.

In rat hepatocyte primary cultures recombinant human interleukin-6 (rhIL-6) induced alpha 2-macroglobulin (alpha 2M) synthesis 54-fold. Half-maximal induction was achieved at a rhIL-6 concentration of 30 pM. RhIL-1 beta led only to a 2-fold increase in alpha 2M synthesis, but strongly impaired the action of IL-6. Intraperitoneal injection of rhIL-6 into male rats resulted in a 19.7-fold increase of alpha 2M mRNA already after 4h. In contrast, alpha 2M mRNA levels (50-fold increase) were reached between 16 and 24 h after intramuscular injection of turpentine. Whereas turpentine-induced inflammation resulted in an increased alpha 2M synthesis in male and female rats, rhIL-6 injection had no effect in female rats. The increases after rhIL-6 administration in mRNA concentrations were followed by corresponding changes in alpha 2M levels in serum. By Northern analysis it was demonstrated that LPS-stimulated human monocytes synthesize IL-6 mRNA. The 5'-end of the rat alpha 2M gene has been isolated and the first 3 exons and 166 base pairs of the 5'-flanking region were identified by a combination of oligonucleotide hybridization and DNA sequencing. The transcriptional start site was determined by RNase protection as well as by primer extension experiments. 5'-CATAAAG-3' and 5'-TCAAAA-3' were found as TATA- and CAAT-box equivalent sequences, respectively. Furthermore, a potential glucocorticoid binding site (5'-TGTTCT-3') was localized on the antisense strand of the alpha 2M gene.

The plasma half life of recombinant human interleukin 1 beta (rhIL 1 beta) was determined in rats by measuring the disappearance of the radioactivity of 125I-labeled rhIL 1 beta from the circulation. The plasma clearance showed a biphasic behavior: an initial fast disappearance (half life of about 3 min) was followed by a second slower one (half life of about 4 h). Twenty minutes after a single-dose injection of 125I-labeled rhIL 1 beta most of the radioactivity was concentrated in kidneys, liver and intestine. rhIL 1 beta induced the synthesis of alpha 1-acid glycoprotein (AGP), alpha 1-cysteine proteinase inhibitor (CPI) and beta-fibrinogen mRNA in liver. Half maximal stimulation was elicited by approximately 3000 U of rhIL 1 beta per animal. The mRNA changes for AGP and CPI were followed by corresponding protein increases in serum. Twenty hours after rhIL 1 beta injection, serum AGP rose from 0.7 to 2.5 mg/ml. CPI increased from 0.3 to 1.9 mg/ml 25 h after administration of rhIL 1 beta. Within 20 h after rhIL 1 beta injection, albumin serum concentration showed a strong decrease, preceded by a reduction in hepatic albumin mRNA levels. Neither changes in albumin synthesis nor degradation can explain this decrease suggesting that other mechanisms such as increased transvascular permeability are involved.