Influence of aeration implements, phosphorus fertilizers, and soil taxa on phosphorus losses from grasslands.

Attenuation of rainfall within the solum may help to move contaminants and nutrients into the soil to be better sequestered or utilized by crops. Surface application of phosphorus (P) amendments to grasslands may lead to elevated concentrations of P in surface runoff and eutrophication of surface waters. Aeration of grasslands has been proposed as a treatment to reduce losses of applied P. Here, results from two small-plot aeration studies and two field-scale, paired-watershed studies are supplemented with previously unpublished soil P data and synthesized. The overall objective of these studies was to determine the impact of aeration on soil P, runoff volume, and runoff P losses from mixed tall fescue [Lolium arundinaceum (Schreb.) Darbysh.]-bermudagrass (Cynodon dactylon L.) grasslands fertilized with P. Small-scale rainfall simulations were conducted on two soil taxa using three types of aeration implements: spikes, disks, and cores. The-field scale study was conducted on four soil taxa with slit and knife aeration. Small-plot studies showed that core aeration reduced loads of total P and dissolved reactive P (DRP) in runoff from plots fertilized with broiler litter and that aeration was effective in reducing P export when it increased soil P in the upper 5 cm. In the field-scale study, slit aeration reduced DRP losses by 35% in fields with well-drained soils but not in poorly drained soils. Flow-weighted concentrations of DRP in aerated fields were related to water-soluble P applied in amendments and soil test P in the upper 5 cm. These studies show that the overall effectiveness of mechanical soil aeration on runoff volume and P losses is controlled by the interaction of soil characteristics such as internal drainage and compaction, soil P, type of surface-applied manure, and type of aeration implement.

Seminal plasma HIV levels in men with asymptomatic sexually transmitted infections.

The effect of asymptomatic sexually transmitted urethral infections on human immunodeficiency virus (HIV) RNA viral load in semen is poorly defined. We studied five such patients. Those on antiretrovirals (n = 2) had lower seminal plasma viral loads (SPVL) (2.11 and 1.98 log(10) copies/mL) than those not on antiretrovirals (n = 3) (2.27-3.78 log(10) copies/mL). One patient who was not taking antiretrovirals had a 94% decline in SPVL after treatment of asymptomatic Chlamydia trachomatis urethritis, suggesting that asymptomatic infection may be a co-factor for HIV transmission.

TNF autovaccination induces self anti-TNF antibodies and inhibits metastasis in a murine melanoma model.

TNF is a proinflammatory cytokine involved in the pathogenesis of chronic inflammatory diseases, but also in metastasis in certain types of cancer. In terms of therapy, TNF is targeted by anti-TNF neutralising monoclonal antibodies or soluble TNF receptors. Recently, a novel strategy based on the generation of self anti-TNF antibodies (TNF autovaccination) has been developed. We have previously shown that TNF autovaccination successfully generates high anti-TNF antibody titres, blocks TNF and ameliorates collagen-induced arthritis in DBA/1 mice. In this study, we examined the ability of TNF autovaccination to generate anti-TNF antibody titres and block metastasis in the murine B16F10 melanoma model. We found that immunisation of C57BL/6 mice with TNF autovaccine produces a 100-fold antibody response to TNF compared to immunisation with phosphate-buffered saline vehicle control and significantly reduces both the number (P<0.01) and size of metastases (P<0.01) of B16F10 melanoma cells. This effect is also observed when an anti-TNF neutralising monoclonal antibody is administered, confirming the essential role TNF plays in metastasis in this model. This study suggests that TNF autovaccination is a cheaper and highly efficient alternative that can block TNF and reduce metastasis in vivo and trials with TNF autovaccination are already underway in patients with metastatic cancer.

Validation of the interleukin-10 knockout mouse model of colitis: antitumour necrosis factor-antibodies suppress the progression of colitis.

Advances in understanding pathogenesis and developing new therapies are hastened by the use of effective animal models of disease. In inflammatory bowel disease, such as Crohn's disease, a variety of models have been used, including the IL-10 knockout mouse. However, in order to be truly valuable, the models need to respond to existing therapy in a way which resembles the human disease. In the light of recent developments, in which refractory Crohn's disease responds well to anti-TNF antibody therapy, we set out to validate the IL-10 knockout model of Crohn's disease by examining its response to anti-TNF therapy. We developed a new scoring system for IL-10 knockout mice, similar to the Crohn's Disease Activity Index in humans, analysed stool samples for cytokines and compared the findings with histology. We found that anti-TNF antibody therapy starting at 4 weeks markedly ameliorated the disease, as judged by the clinical score or by histological analysis of the gut. Furthermore, analysis of stool samples for cytokines revealed a marked diminution of inflammatory cytokines, adding a further accurate measure of the improvement. This model may thus be useful for evaluating other therapeutic modalities of relevance to Crohn's disease.

Increased production of intracellular interleukin-1 receptor antagonist type I in the synovium of mice with collagen-induced arthritis: a possible role in the resolution of arthritis.

OBJECTIVE: To examine the patterns of production of interleukin-1 receptor antagonist (IL-1Ra) isoforms and of IL-1beta during arthritis in vivo. METHODS: Arthritis was induced in DBA/1 mice by immunization with type II collagen, and the production of IL-1Ra isoforms was examined in whole joints and in dissected synovial tissues by reverse transcription-polymerase chain reaction (RT-PCR), RNase protection assay, Western blotting, immunostaining, and in situ hybridization. Production of IL-1beta also was examined using similar approaches. RESULTS: Production of IL-1Ra increased in the joints during collagen-induced arthritis (CIA). By RT-PCR, secreted IL-1Ra messenger RNA (mRNA) was detected in normal joints, whereas intracellular IL-1Ra type I (icIL-1Ra1) mRNA was only produced in inflamed joints. Western blot studies showed that icIL-1Ra1 protein levels increased in the joints during the course of CIA and that icIL-1Ra3 protein was also present in low amounts. RNase protection assays showed that the IL-1beta:IL-1Ra mRNA ratio was increased in inflamed joints through day 14 of arthritis, whereas a reverse pattern was present at later time points (from day 20 to day 60). Consistent with this finding, immunohistochemistry and in situ hybridization studies confirmed that icIL-1Ra1 was only present in inflamed joints. The histologic evaluation of CIA during the course of the disease indicated a resolution of acute inflammation, since icIL-1Ra1 production increased and the ratio of IL-1beta to total IL-1Ra decreased. CONCLUSION: Production of IL-1Ra isoforms, particularly icIL-1Ra1, is stimulated in inflamed joints during CIA in mice. The combination of decreased production of IL-1beta and elevated levels of icIL-1Ra1 during the course of CIA was associated with a reduction in inflammatory activity. These results suggest that icIL-1Ra1 may play a role in the resolution of murine CIA.

Combination therapy with DMARDs and biological agents in collagen-induced arthritis.

There is increasing interest in the use of combination therapy for rheumatoid arthritis and in the possibility of combining the conventional drug approach with newer biological therapies. Animal models of arthritis provide important tools for evaluating novel forms of therapy and for eludicating mechanisms of drug action. In this paper, we review the results of our own research into combination therapy in collagen-induced arthritis using biological therapies such as anti-tumor necrosis factor alpha, anti-CD4, and anti-interleukin 12 monoclonal antibodies, and small molecular weight compounds such as cyclosporin and the phosphodiesterase IV (PDE IV) inhibitor rolipram.

Release of Ca(2+) from intracellular stores and entry of extracellular Ca(2+) are involved in sea squirt sperm activation.

A rise in intracellular free Ca(2+) concentration ([Ca(2+)](i)) is required to activate sperm of all organisms studied. Such elevation of [Ca(2+)](i) can occur either by influx of extracellular Ca(2+) or by release of Ca(2+) from intracellular stores. We have examined these sources of Ca(2+) in sperm from the sea squirt Ascidia ceratodes using mitochondrial translocation to evaluate activation and the Ca(2+)-sensitive dye fura-2 to monitor [Ca(2+)](i) by bulk spectrofluorometry. Sperm activation artificially evoked by incubation in high-pH seawater was inhibited by reducing seawater [Ca(2+)], as well as by the presence of high [K(+)](o) or the Ca channel blockers pimozide, penfluridol, or Ni(2+), but not nifedipine or Co(2+). The accompanying rise in [Ca(2+)](i) was also blocked by pimozide or penfluridol. These results indicate that activation produced by alkaline incubation involves opening of plasmalemmal voltage-dependent Ca channels and Ca(2+) entry to initiate mitochondrial translocation. Incubation in thimerosal or thapsigargin, but not ryanodine (even if combined with caffeine pretreatment), evoked sperm activation. Activation by thimerosal was insensitive to reduced external calcium and to Ca channel blockers. Sperm [Ca(2+)](i) increased upon incubation in high-pH or thimerosal-containing seawater, but only the high-pH-dependent elevation in [Ca(2+)](i) could be inhibited by pimozide or penfluridol. Treatment with the protonophore CCCP indicated that only a small percentage of sperm could release enough Ca(2+) from mitochondria to cause activation. Inositol 1,4,5-trisphosphate (IP(3)) delivered by liposomes or by permeabilization increased sperm activation. Both of these effects were blocked by heparin. We conclude that high external pH induces intracellular alkalization that directly or indirectly activates plasma membrane voltage-dependent Ca channels allowing entry of external Ca(2+) and that thimerosal stimulates release of Ca(2+) from IP(3)-sensitive intracellular stores.

Paradoxical effects of adenovirus-mediated blockade of TNF activity in murine collagen-induced arthritis.

Collagen-induced arthritis (CIA) is an experimental model of arthritis widely used to dissect the pathogenesis of human rheumatoid arthritis and to identify potential therapeutic targets. Among these, TNF-alpha has been recognized to play an important role. Here we investigate the feasibility and therapeutic efficacy of prolonged blockade of TNF-alpha activity through the adenovirus-mediated gene delivery of a dimeric chimeric human p55 TNFR-IgG fusion protein and compare it to protein therapy in established CIA. A single i.v. administration of the replication-deficient adenovirus yielded microgram serum levels of the chimeric fusion protein and ameliorated CIA for 10 days. Subsequently, benefit was lost and a rebound to greater inflammatory activity was observed despite the continual presence of bioactive TNFR fusion protein. A similar trend was also observed in mice injected directly with comparable amounts of a human TNFR-IgG fusion protein, whereas the administration of a control adenovirus-encoding beta-galactosidase or of a control human IgG1 protein did not significantly affect the disease course. The mechanisms of the rebound of CIA were investigated, and augmented Ab response to collagen type II and TNFR were identified as potential causes. Our results confirm the feasibility of adenovirus-mediated gene delivery of cytokine inhibitors in animal models of autoimmune diseases for investigational purposes and highlight the importance of prolonged studies. Further investigations are needed to optimize ways of exploiting the potential of adenoviral gene therapy in RA.

Therapeutic antibodies elicited by immunization against TNF-alpha.

Tumor necrosis factor-alpha (TNF-alpha) is critically involved in the pathogenesis of several chronic inflammatory diseases. Monoclonal antibodies against TNF-alpha are currently used for the treatment of rheumatoid arthritis and Crohn's disease. This report describes a simple and effective method for active immunization against self TNF-alpha. This vaccination approach leads to a T-cell-dependent polyclonal and sustainable anti-TNF-alpha autoantibody response that declines upon discontinuation of booster injections. The autoantibodies are elicited by injecting modified recombinant TNF-alpha molecules containing foreign immunodominant T-helper epitopes. In mice immunized with such molecules, the symptoms of experimental cachexia and type II collagen-induced arthritis are ameliorated. These results suggest that vaccination against TNF-alpha may be a useful approach for the treatment of rheumatoid arthritis and other chronic inflammatory diseases.

Anti-IL-12 and anti-TNF antibodies synergistically suppress the progression of murine collagen-induced arthritis.

The co-ordinate role of the Th1 cytokine IL-12 and the proinflammatory cytokine TNF in arthritis was explored using the DBA/1 mouse model, collagen-induced arthritis (CIA). In this study, mice with established arthritis were treated with anti-IL-12 and/or anti-TNF antibodies for 10 days from the onset of disease. Clinical assessment showed that the combined antibody treatment ameliorated disease severity to a greater extent than anti-TNF alone. Supporting these observations, histological analysis revealed that there was a reduced joint damage in the mice that received combined anti-IL-12 and anti-TNF treatment, compared to the other treatment groups. Anti-IL-12 had no statistically significant effect on the clinical outcome of disease. The combination of anti-IL-12 and anti-TNF treatment was found to reduce collagen type II (CII)-specific lymph node cell IFN-gamma production and proliferation, as well as decrease the anti-CII IgG2a:IgG1 ratio more effectively than either treatment alone. When the antibodies were added to synovial cells from arthritic mice and bone marrow macrophages in vitro, anti-TNF diminished IL-12 production, but anti-IL-12 had no effect on TNF production. These data suggest that, through the partial regulation of IL-12, TNF modulates the immune response in arthritis, as well as the inflammatory response. The synergistic action of anti-TNF and anti-IL-12 on CIA may provide a new therapeutic approach for treating rheumatoid arthritis.

The beta2-adrenergic agonist salbutamol is a potent suppressor of established collagen-induced arthritis: mechanisms of action.

The therapeutic potential of salbutamol, a beta2-adrenergic agonist, was explored in collagen-induced arthritis. This study was based on a report that salbutamol, by elevating intracellular cAMP, inhibits IL-12 production by macrophages and dendritic cells, thus preventing Th1 development. Ten-week-old male DBA/1 mice were immunized by intradermal injection of type II collagen in CFA. Arthritis developed 15-30 days later and the mice were treated after onset of disease with salbutamol, 200 microgram i.p. After 10 days, the mice were sacrificed, and the hind paws were evaluated histologically. Salbutamol, 200 microgram daily or every other day, had a profound therapeutic effect on the clinical progression of arthritis, as assessed by clinical score and paw thickness. The therapeutic effect was dose dependent. Daily administration of 200 microgram of salbutamol offered the best protection against joint damage, as assessed by histology. In vitro, salbutamol reduced IL-12 and TNF-alpha release by peritoneal macrophages in a dose-dependent manner, as well as TNF release by synovial cells from arthritic mice. Ex vivo, draining lymph node cells of the salbutamol-treated arthritic mice showed a diminished CII-specific IFN-gamma production and proliferation. In vivo, salbutamol specifically blocked mast cell degranulation in joint tissues. In conclusion, salbutamol has important effects on the immunoinflammatory response and a significant therapeutic action in collagen-induced arthritis.

An anti-inflammatory role for interleukin-11 in established murine collagen-induced arthritis.

Interleukin-11 (IL-11) is a cytokine belonging to the IL-6 family which has both pro- and anti-inflammatory potential. Like IL-6 it can diminish tumour necrosis factor-alpha and IL-1 production, and augment immunoglobulin synthesis. We have explored the immunomodulatory effects of IL-11 treatment in mice in a model of inflammatory autoimmune joint disease, collagen-induced arthritis (CIA). Recombinant human IL-11 was administered at various doses to DBA/1 mice after the onset of CIA. IL-11 treatment caused a significant reduction in the clinical severity of established CIA, which was associated with protection from joint damage, as assessed by histology. Although there was a suggestion at high doses of IL-11 that the anticollagen type II (CII) response may have been augmented, there was no statistically significant effect of IL-11 treatment on anti-CII antibody levels. Similarly, the acute-phase reactant serum amyloid P was only elevated in mice receiving very high doses (50-100 microgram/day) of IL-11. Endogenous IL-11 was abundantly produced in synovial membrane cultures derived from CII-immunized mice with active disease, suggesting that, as in rheumatoid arthritis, this cytokine is spontaneously produced in the inflammatory response in CIA. The results presented here demonstrate an anti-arthritic immunoregulatory role for IL-11 in murine CIA, and suggest that IL-11 is a candidate therapeutic molecule for human inflammatory arthritic diseases.

Potassium current development and its linkage to membrane expansion during growth of cultured embryonic mouse hippocampal neurons: sensitivity to inhibitors of phosphatidylinositol 3-kinase and other protein kinases.

Hippocampal pyramidal neurons express three major voltage-dependent potassium currents, IA, ID, and IK. During hippocampal development, IA, the rapidly activating and inactivating transient potassium current, is detected soon after pyramidal neurons can be morphologically identified. Appearance of IA in developing pyramidal neurons is dependent on contact with cocultured astroglial cells; cultured pyramidal neurons not in contact with astroglial cells have reduced membrane area and IA (Wu and Barish, 1994). We have examined intracellular signaling pathways that could contribute to the regulation of IA development by probing developing pyramidal neurons with kinase inhibitors. We observed that exposure to LY294002 or wortmannin, inhibitors of phosphatidylinositol (PI) 3-kinase, reduced somatic cross-sectional area, neurite outgrowth, whole-cell capacitance, IA amplitude and density (amplitude normalized to membrane area), and immunoreactivity for Kv4.2 and/or Kv4.3 (potassium channel subunits likely to be present in the channels carrying IA). In contrast, exposure to ML-9 or KN-62, inhibitors of myosin light chain kinase or Ca2+-calmodulin-dependent protein kinase II (CaMKII), reduced membrane area and IA amplitude but did not affect IA density or Kv4. 2/3 immunoreactivity to the same extent as inhibitors of PI 3-kinase. Unexpectedly, exposure to bisindolymaleimide I or calphostin C, inhibitors of protein kinase C (PKC), did not affect membrane area or potassium current development. Our data suggest that PI 3-kinases regulate both A-type potassium channel synthesis and plasmalemmal insertion of vesicles bearing these potassium channels. CaMKII appears to regulate fusion of channel-bearing vesicles with the plasmalemma and myosin light chain kinase to regulate centripetal transport of channel-bearing vesicles from the Golgi. We further suggest that astroglial cells exert their influence on pyramidal neuron development through activation of PI 3-kinases.

Blockade of IL-12 during the induction of collagen-induced arthritis (CIA) markedly attenuates the severity of the arthritis.

The effect of blocking IL-12, a potent inducer of interferon-gamma (IFN-gamma) and promoter of Th1 cell responses, during the induction phase of CIA was investigated. Arthritis was elicited in male DBA/1 mice by immunizing with type II collagen (CII) in Freund's complete adjuvant. Neutralizing anti-IL-12 antibodies were administered twice weekly from CII immunization. It was found that administration of anti-IL-12 from immunization until the onset of clinical arthritis did not lower the incidence of arthritis, but dramatically attenuated the severity of the disease, both clinically and histopathologically. This regime was associated with reduced IFN-gamma levels produced by ex vivo CII-stimulated draining lymph node cells, and with diminished spontaneous ex vivo production of tumour necrosis factor (TNF), IL-6 and IL-10 by freshly isolated synovial cells. Total anti-CII antibody serum levels in these mice were lower than in the controls, but there was no change in the IgG2a/IgG1 ratio. These findings confirm that IL-12 has a major role in the induction of murine CIA and suggests that this disease is propagated, in part, by cells of the Th1 phenotype.

Mouse brain potassium channel beta1 subunit mRNA: cloning and distribution during development.

The pore-forming alpha subunits of voltage-gated potassium channels in neurons and other excitable cells are expressed in association with accessory beta subunits. These subunits both promote insertion of channel complexes into surface membranes and influence their electrophysiological properties. As part of an effort to understand the regulation of voltage-gated potassium channels during development, we cloned the mouse homolog of the rat Kvbeta1 potassium channel subunit. Kvbeta1 subunits are known to associate preferentially with Shaker (Kv1)-related alpha subunits. We then used a digoxigenin-tagged cRNA probe and in situ hybridization techniques to visualize the appearance of Kvbeta1 mRNA transcripts during late embryonic and early neonatal development of the mouse brain. We detected Kvbeta1-specific labeling of cells in hippocampus, cerebral cortex, caudate putamen, colliculus, and cerebellum. In hippocampus, we observed Kvbeta1 mRNA in CA3 pyramidal neurons at the earliest time examined, embryonic day 16 (E16). Between E16 and postnatal day 7 (P7), cell labeling increased uniformly across the pyramidal neurons of Ammon's horn (CA1, CA2, and CA3). Subsequently, between P7 and P22, regional differences characteristic of mature hippocampus appeared-intense labeling of neurons in CA3 and CA1, and less in CA2. In cortex, labeling of cells in the subplate and cortical plate layers was observed at E16. During development, the intensity of this labeling increased, and labeled cells persisted into the adult stage in the deep cortical layer (VIb) formed from subplate neurons. Additional labeling of scattered solitary cells in cortical layers II-VIa emerged between P3 and P7 and was prominent in mature cortex. In caudate putamen, Kvbeta1-labeled cells were observed at P1 and were restricted to the lateral and rostral half of the caudate. During development, labeling expanded caudally and medially and eventually filled the mature caudate putamen. In colliculus, a small population of inferior colliculus cells showed labeling at P7, and additional labeling of scattered cells appeared during development. In superior colliculus, labeling was observed only in the adult deep gray layer. In cerebellum, intense labeling was observed in Purkinje cells at all stages between P1 and adult. Labeling was also seen in granule neurons in the external granule layer at early postnatal stages and in the inner granule layer beginning at P7.

DBA/1 mice expressing the human TNF-alpha transgene develop a severe, erosive arthritis: characterization of the cytokine cascade and cellular composition.

Arthritis spontaneously develops in mice expressing a human TNF-alpha transgene modified with the 3' untranslated region of beta-globin. We have backcrossed these mice onto the arthritis-susceptible DBA/1 background and found an acceleration of the onset of arthritis with successive generations of interbreeding. Bioactive TNF-alpha in primary synovial membrane cell cultures was significantly higher in the DBA/1 transgenic mice than in transgenic mice on the original background. Elevated levels of human TNF-alpha were accompanied by increases in synovial cell expression of murine IL-1beta and IL-6, but murine granulocyte-macrophage CSF, IFN-gamma, and IL-4 could not be detected. Interestingly, the anti-inflammatory cytokine IL-10 could be detected, but levels were not modulated by expression of the transgene. Analysis of the synovial membrane cell composition revealed that >50% of synovial cells were CD45-negative cells, presumably fibroblasts and endothelial cells, and the majority of CD45-expressing cells were neutrophils. Peritoneal macrophages and lymphocytes from the spleen, bone marrow, and lymph nodes required LPS stimulation to produce human TNF-alpha, indicating that, when activated, cells of these lineages were capable of expressing the transgene; however, few were found in synovial tissues. In contrast, fibroblasts derived from synovial tissue spontaneously released human TNF-alpha, and using immunohistochemical techniques, this cytokine was localized to fibroblast-like cells and chondrocytes. We propose that arthritis in DBA/1 human TNF-alpha transgenic mice is driven in part through the spontaneous expression of transgene by connective tissue cells, and there is little evidence of the participation of lymphocytes in this model.

Suppression of collagen-induced arthritis by continuous administration of IL-4.

The onset of collagen-induced arthritis in DBA/1 mice is accompanied by a predominantly Th1 response, characterized by production of the proinflammatory cytokines IFN-gamma and TNF-alpha, and a predominance of IgG2a anti-collagen Abs. This study has primarily addressed the effects of continuous administration of exogenous IL-4, a Th2 cytokine, on collagen-induced arthritis in terms of time of onset, clinical symptoms, and histologic changes compared with those in untreated controls. The contributions of Th1 and Th2 cell responses were studied by examining anti-CII IgG subclasses, serum IgE levels, and cytokine production by synovial membrane and lymph node cell cultures. Continuous exposure to IL-4 for 28 days significantly delayed the onset of arthritis from 19 to 37 days and suppressed clinical symptoms. Arthritis occurred approximately 13 to 24 days after treatment ceased. Thereafter, the severity and duration of clinical symptoms were similar to those in control animals, although both joint damage and inflammation at the histologic and cellular levels were less severe than those in untreated controls. During IL-4 treatment, anti-collagen Ab levels were reduced (most significantly those of the IgG2a subclass), histology scores were lower, and the most striking effect was a 1000-fold decrease in TNF-alpha secretion by synovial cells. No significant differences in IgE levels were found between controls and IL-4-treated mice. These data suggest that the anti-inflammatory properties of IL-4 are mediated in part by down-regulation of Th1 responses rather than up-regulation of Th2 responses.

Modulation of proinflammatory cytokine release in rheumatoid synovial membrane cell cultures. Comparison of monoclonal anti TNF-alpha antibody with the interleukin-1 receptor antagonist.

While there is an extensive literature on cytokine regulation in vivo using human cell lines or peripheral blood monocytes, very little is known about cytokine regulation within the multicellular environment of inflammatory sites in vivo. We have previously shown that in rheumatoid synovial membrane cultures, a complex, but pathophysiologically relevant mixture of cells, the addition of a neutralizing anti TNF-alpha antibody inhibits the production of IL-1 and GM-CSF, indicating the presence of a cytokine 'cascade' in this inflammatory tissue. In this paper we demonstrate that the interactivities between cytokines in rheumatoid arthritis also extends to other cytokines, such as IL-6 and IL-8, and that within the IL-1 family it is IL-1 beta in particular which is downregulated by neutralizing TNF-alpha activity. The cytokine interactions are unidirectional, in that neutralization of TNF-alpha reduced IL-1 beta, IL-6 and IL-8 production, whereas treatment of the rheumatoid synovial membrane cells with a neutralizing concentration of the IL-1 receptor antagonist (IL-1ra) reduced IL-6 and IL-8 production but not TNF-alpha production. These results suggest a rationale for the profound anti-inflammatory effects and consequent clinical benefit noted in RA patients treated recently in clinical trials with a chimeric anti-TNF-alpha antibody in vivo.

TNF receptor fusion proteins are effective inhibitors of TNF-mediated cytotoxicity on human KYM-1D4 rhabdomyosarcoma cells.

KYM-1D4 cells are a subline derived from a human rhabdomyosarcoma which are highly sensitive to TNF-mediated cytotoxicity. They were selected for this study because they express human TNF-R and are therefore a more relevant target for comparing the potential therapeutic value of human TNF-inhibitory agents than the usual murine cell lines. Two recombinant soluble TNF-R-IgG fusion proteins, one containing p55 TNR-R, the other containing p75 TNF-R, and a recombinant monomeric soluble p55 TNF-R were all found to block the cytotoxicity generated by human TNF-alpha and LT as well as also murine TNF. The p55 TNF-R-IgG fusion protein (p55-sf2) was the most effective of the antagonists tested, requiring an equimolar, (based on a monomeric configuration of TNF-alpha) or a 3-fold higher (based on a trimeric configuration of TNF-alpha) molar concentration to inhibit the cytotoxicity mediated TNF-alpha by 50%. p55-sf2 was also as effective at inhibiting the cytotoxicity mediated by LT or murine TNF in the KYM-1D4 assay. In contrast, the monomeric soluble p55 TNF-R was the least effective inhibitor, requiring a > 4000-fold higher molar concentration than p55-sf2 to achieve a similar degree of protection. The fusion proteins, particularly p55-sf2, may be useful as human therapeutic agents, as at low concentrations they can prevent both TNF-alpha-mediated and LT-mediated effects on human cells. As TNF-R-IgG fusion proteins also block the action of murine TNF in vitro, they may also be useful in the investigation of murine models of human inflammatory disease.

p55 and p75 tumor necrosis factor receptors are expressed and mediate common functions in synovial fibroblasts and other fibroblasts.

There is increasing evidence that TNF-alpha is a cytokine of major importance in the pathogenesis of rheumatoid arthritis. Since TNF-alpha mediates its effects via high affinity receptors, we were interested in investigating their expression and function in cells from rheumatoid tissue. Synovial fibroblasts derived from rheumatoid synovial tissue are stimulated by TNF-alpha to proliferate and release cytokines, prostaglandins, proteases and protease inhibitors. We have evaluated through which receptor stimulation of DNA synthesis and the release of the proinflammatory agents, IL-6, IL-8 and PGE2 are induced. It was found that rheumatoid synovial fibroblasts express both the p55 and p75 TNF receptor, in a ratio of 4:1. TNF-alpha-stimulated synovial fibroblast DNA synthesis and the release of IL-6, IL-8 and PGE2 was inhibited by antagonist monoclonal antibodies against either the p55 or the p75 TNF receptor, although the blockade of the p55 TNF receptor had a more potent effect than inhibition of the p75 TNF receptor alone. Similarly, specific monoclonal antibodies, agonistic for either the p55 or p75 TNF receptor stimulated synovial fibroblast DNA synthesis, as well as IL-6, IL-8 and PGE2 release. Both p55 and p75 TNF receptors on dermal and gingival fibroblasts were also involved in TNF-alpha-mediated DNA synthesis and IL-6, IL-8 and PGE2 release, although differences in the levels of DNA synthesis and release of inflammatory cytokines and PGE2 were observed between the three fibroblast types.