Comparative analysis of transcription and protein release of the inflammatory cytokines interleukin-1 beta (IL-1 beta) and interleukin-8 (IL-8) following major burn and mechanical trauma.

The precondition for the systematic modulation of host impairing behavior of hyperactivated monocytes following trauma is to fully understand the mechanistic basis of cellular dysfunction. It was the objective of this study to scrutinize the synthesis patterns and the level of regulation of the functionally related inflammatory cytokines interleukin (IL)-1 beta and IL-8 under stressful conditions. We compared the quantity of cytokine protein release in lipopolysaccharide-stimulated in vitro cultures of peripheral blood mononuclear leukocytes with the signal intensity of the corresponding detectable mRNAs. Fourteen patients with major burn or multiple trauma on consecutive days post-trauma and healthy volunteers were studied. We saw an almost identical pattern of synthesis for both monokines during the time of observation, with a considerable impairment until day 5 post-trauma and recovery thereafter. In contrast to IL-1 beta, a clear concurrence between mRNA signal intensity and the quantity of protein release was found in the majority of patients for IL-8. From these data we conclude that the launching mechanisms for the de novo synthesis for both monokines under stress differ greatly, with IL-8 being clearly regulated on the transcriptional level, whereas the downregulation of IL-1 beta occurs, most likely, on the post-transcriptional level.

CGP 47969A: a novel inhibitor of the synthesis of inflammatory cytokines.

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.

Interleukin-1 (IL-1) production in a mouse tissue chamber model of inflammation. I. Development and initial characterisation of the model.

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.

Interleukin-1 (IL-1) production in a mouse tissue chamber model of inflammation. II. Identification of (tissue) macrophages as the IL-1 producing cells and the effect of anti-inflammatory drugs.

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.

Measurement of interleukin 1 alpha and 1 beta (IL-1 alpha and IL-1 beta) in human cystic lesions of the jaw. Implications for the pathogenesis of radicular cysts.

Human radicular cystic tissue of jaws was found to contain between 0.823 pg/mg to 18.026 pg/mg interleukin 1 beta and from 0.34 pg/mg to 0.708 pg/mg interleukin 1 alpha. No IL-1 beta and alpha could be found in specimens from healthy patients. A finding which may be extremely relevant in cystic growth and episodes of alveolar bone resorption around the cystic lesion.

IGG-stimulated and LPS-stimulated monocytes elaborate transforming growth factor type beta (TGF-beta) in active form.

Mononuclear cells (MNC) stimulated either with lipopolysaccharide (LPS) or with surface-adsorbed IgG elaborated significant amounts of tumor necrosis factor (TNF) bioactivity, as well as immunoenzymatically detectable TNF-alpha and interleukin-1 beta. (IL1-beta). In contrast, IgG-stimulated cells released little IL1 bioactivity, but released an IL1 inhibitor, as determined by the thymocyte costimulatory assay (LAF assay). This inhibition was not due to an inhibitory effect of cyclooxygenase products, e.g. prostaglandin-E2 in the LAF assay. In contrast, antibodies against transforming growth factor type beta (TGF-beta), which is an important inhibitor of the LAF assay, augmented the LAF activity of supernatants from LPS-stimulated and IgG-stimulated MNC. Anti-TGF-beta-modulated LAF inhibition was enhanced by acid treatment of supernatants from mononuclear cells, but not of those from purified monocytes. Antibody blocking experiments point for the first time to a TGF-beta species other than type 1 as a monocyte-derived TGF-beta activity. Thus, TGF-beta released in active form from monocytes may be the more important antagonist of IL1 than cyclooxygenase-derived mediators. It implies that the LAF assay, in the absence of anti-TGF-beta antibodies, is an inadequate indicator of IL1 activity.

Increased interleukin-1 beta (IL-1 beta) concentration in gingival tissue from periodontitis patients.

Human gingival tissues from periodontitis patients were found to contain from 126 fg/mg to 2161 fg/mg interleukin-1 beta as determined by a sensitive enzyme linked immunoassay. No IL-1 beta could be found in normal gingival tissue. This finding may have important consequences relevant to connective tissue destruction and episodes of alveolar bone resorption characteristic of chronic periodontitis.

An assay for the detection of interleukin-1 synthesis inhibitors: effects of antirheumatic drugs.

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.

Differential immunological response of patients with rheumatoid arthritis towards two different Epstein-Barr virus strains: inhibition of interleukin-1 release by the B95-8, but not the P3HR-1 virus strain.

An abnormal immune response towards the Epstein-Barr virus (EBV) has been documented in patients with rheumatoid arthritis (RA). To investigate whether these findings are due to the transformation event caused by EBV, RA blood mononuclear cells and monocyte-enriched preparations were incubated with two different EBV strains: the transforming virus secreted by the cell line B95-8 and the virus released by the P3HR-1 cell line that is not able to transform due to a small deletion in the U2 region of the virus genome. Immunological response was determined by the production of interleukin-1 (IL-1) and tumor necrosis factor (TNF) using ELISA and bioassay systems. There was a striking difference in cytokine measurements with a strong inhibition of IL-1 and TNF production after incubation with the B95-8 virus, but not the P3HR-1 virus. These data indicate that the disturbed reaction of the immune system towards EBV is either dependent on the full transformation of B cells in RA patients or alternatively due to the secretion of a cytokine inhibitor by the B95-8 cell line.

Arthritis induced by continuous infusion of hr-interleukin-1 alpha into the rabbit knee-joint.

Continuous infusion of 200 ng/day hrIL-1 alpha for 14 days into knee-joints of rabbits leads to a severe arthritis of low aggressivity. This arthritis shows simultaneously characteristics of acute (serous and fibrinous exudation, polymorph infiltration, etc.) as well as chronic (synovial cell proliferation and fibrosis, pannus formation, cartilage and bone erosion, etc.) inflammation. The arthritis was associated with a distinct loss of metachromasia of the articular cartilage. These results indicate that IL-1 might play an important role in the induction and maintenance of arthritis.

Binding characteristics of human serum amyloid P component.

Serum amyloid P component (SAP), a normal human plasma glycoprotein, was found in a solid phase ELISA to have Ca2+-dependent binding for keyhole limpet haemocyanin (KLH), pectic acid, trinitrophenylated (TNP) macromolecules, and plastic surfaces. The binding to TNP-KLH was used to develop a sensitive ELISA. The binding of SAP to the ligands mentioned was inhibited by EDTA, KLH, pectic acid, TNP-conjugated macromolecules (bovine serum albumin, polyacrylhydrazide), and p-nitrophenylarsonic acid. Underivatized and DNP-conjugated macromolecules did not inhibit the SAP binding; arsenilic acid, picric acid, and dinitrophenyl were weak inhibitors. SAP bound to TNP-agarose was eluated by either EDTA or p-nitrophenylarsonic acid. Thus, a unique region of SAP is responsible for the polyspecific binding. We suggest that the polyspecific binding of SAP takes place through a Ca2+ bridge: half of the metal coordination sphere is occupied by SAP, with the other half available to interact with metal ligand.

Quantitation of serum amyloid P component by an enzyme-linked immunoassay.

A solid phase enzyme-linked immunoassay (ELISA) for the murine acute-phase reactant, serum amyloid P component (SAP), was developed. The assay is based on our finding of a calcium-dependent binding of SAP to trinitrophenyl-conjugated proteins. The wells of polystyrene microtiter plates are coated with trinitrophenylated keyhole limpet hemocyanin (TNP-KLH), then incubated with SAP-containing samples. The amount of SAP is determined by indirect ELISA, wells being sequentially incubated with rabbit anti-SAP antiserum and horseradish peroxidase-linked donkey anti-rabbit IgG conjugate. The limit of sensitivity of the assay is 0.6 ng/ml SAP. Comparison with data on sera obtained by rocket immunoelectrophoresis assay yielded a correlation coefficient of 0.89. The binding of SAP to TNP-KLH was inhibited by calcium chelators and low concentrations of non-ionic detergents. Chromatography of serum on TNP-Sepharose provided a efficient and simple way of purifying SAP. The assay was also adapted for the quantitation of human SAP. The use of the assay in studying the binding specificities of SAP and its physiological role is discussed.

Murine polyspecific antibodies. I. Monoclonal and serum anti-DNA antibodies cross-reactive with 2,4,6-trinitrophenyl derivatives.

Six anti-DNA hybridoma autoantibodies were prepared by fusing spleen cells from unimmunized MRL/MpJ/lpr/lpr female mice with BALB/c myeloma cells. The monoclonal antibodies were analyzed by solid-phase ELISA for antigen-binding specificities. Three antibodies (62A2, 85A5, and 43B2) bound ssDNA, TNP-KLH, and recognized an epitope(s) present on insolubilized proteins such as BSA, KLH, ferritin, and insulin. The antibodies bound, with a marked preference, TNP-KLH, either soluble or insoluble. The other three antibodies (35A1, 32C5, and 39D2) bound only ssDNA. However, this binding was inhibited by free flavinic acid. None of the six antibodies bound either cardiolipin or proteoglycans, indicating that they do not recognize the repeating negatively charge units common to cardiolipin, proteoglycans, and DNA. All six monoclonal antibodies were purified by affinity chromatography with TNP-Sepharose. Moreover, both anti-DNA and anti-TNP antibodies from sera of nonautoimmune and autoimmune mice were purified easily on TNP-Sepharose.

Serum amyloid P component and autoimmune parameters in the assessment of arthritis activity in MRL/lpr/lpr mice.

The concentration of immunoglobulins, anti-ssDNA, anti-dsDNA, anti-TNP antibodies, IgM rheumatoid factor, C3, immune complexes and serum amyloid P component (SAP), in the serum were measured in 68 male and female MRL/lpr/lpr mice, a strain affected with a systemic autoimmune disease. The degree of lymphoproliferation was assessed by the spleen weight. Spontaneous secretion of immunoglobulins and anti dsDNA antibodies were measured in spleen cell cultures. All mice presented age related increases or decreases (C3) in the level of measured parameters. Inflammatory lesions were detected, by light microscopy in the joints of all mice. There was a significant correlation, in both sexes between the serum level of SAP and the severity of the polyarthritis, as assessed by light microscopy. In female mice the levels of anti-dsDNA antibodies, immunoglobulins, either measured in serum or in the spleen compartment, and circulating immune complexes also showed correlation with the activity of the arthritis, but neither of these variables correlated as closely with arthritis scores as did serum SAP.

Polyarthritis in MRL lpr/lpr mice.

Mice of the inbred strain MRL/MpJ-lpr/lpr are affected by a systemic autoimmune disease and a spontaneously occurring polyarthritis. To characterize the arthritis a histopathological study was performed on the joints of the four limbs and of the spinal column of 7, 16, 22 and 28-week-old animals of both sexes. Polyarthritis, the severity of which increased with age was detected in all mice. Proliferation of the synovial lining cells, already evident in 7-week-old animals, was the initial lesion. In the majority of cases infiltrates containing lymphocytes with a few plasmocytes, histiocytes, polymorphonuclear neutrophils and eosinophils were detected later on. The most pronounced changes were observed in the hind-paws, the fore-paws, the knee and hip joints, paired articulations being symmetrically involved. A pannus was seen at the most in 10% of the joints leading to limited and superficial destruction of the cartilage. Rheumatoid nodules were not seen. From 16 weeks of age deposits of unknown nature, often surrounded by phagocytosing macrophages and/or neutrophils, were observed in the articular and/or extra-articular connective tissue and in the vessels. There was a positive correlation between their presence and the intensity of the arthritis. The articular lesions in our study differ from those in rheumatoid arthritis because they lacked the specific and characteristic histological features of the human disease.