Phosphodiesterase 4 (PDE4) regulation of proinflammatory cytokine and chemokine release from rheumatoid synovial membrane.

Background: The cAMP-metabolising enzyme, phosphodiesterase 4 (PDE4), has been implicated in a number of immune responses, including tumour necrosis factor α (TNFα) production. To date, few data have directly addressed whether synovial cytokine and chemokine production is modified by PDE4.

Objective: Using specific PDE4 inhibitors, roflumilast plus two novel inhibitors, INH 0061 and INH 0062, the authors studied the effect of PDE4 inhibition on proinflammatory cytokine and chemokine release from primary rheumatoid arthritis (RA) synovial digest suspensions and in a macrophage T cell co-culture assay system.

Gene expression profiles in the rat streptococcal cell wall-induced arthritis model identified using microarray analysis.

Experimental arthritis models are considered valuable tools for delineating mechanisms of inflammation and autoimmune phenomena. Use of microarray-based methods represents a new and challenging approach that allows molecular dissection of complex autoimmune diseases such as arthritis. In order to characterize the temporal gene expression profile in joints from the reactivation model of streptococcal cell wall (SCW)-induced arthritis in Lewis (LEW/N) rats, total RNA was extracted from ankle joints from naive, SCW injected, or phosphate buffered saline injected animals (time course study) and gene expression was analyzed using Affymetrix oligonucleotide microarray technology (RAE230A).

Characterization of pristane-induced arthritis, a murine model of chronic disease: response to antirheumatic agents, expression of joint cytokines, and immunopathology.

Objective: To characterize chronic murine pristane-induced arthritis (PIA) with regard to the response to antirheumatic agents, expression levels of proinflammatory cytokines, and immunopathologic features.

Methods: Male DBA/1 mice were injected intraperitoneally with pristane oil to induce a chronic polyarthritis, which was monitored by visual scoring. Serum antibody and splenocyte responses to a panel of putative joint-derived autoantigens were measured.

Joint cytokine quantification in two rodent arthritis models: kinetics of expression, correlation of mRNA and protein levels and response to prednisolone treatment.

Biomarker quantification in disease tissues from animal models of rheumatoid arthritis (RA) can help to provide insights into the mechanisms of action of novel therapeutic agents. In this study we validated the kinetics of IL-1beta, TNF-alpha and IL-6 mRNA and protein expression levels in joints from DBA/1OlaHsd murine collagen-induced arthritis (CIA) and Lewis rat Streptococcal cell wall (SCW)-induced arthritis by real-time polymerase chain reaction (PCR) TaqMan and Enzyme-linked immunosorbent assay (ELISA). Prednisolone was used as a reference to investigate any correlation between clinical response and cytokine levels at selected time-points.

Interleukin 17 induces cartilage collagen breakdown: novel synergistic effects in combination with proinflammatory cytokines.

Objective: To investigate whether interleukin 17 (IL17), derived specifically from T cells, can promote type II collagen release from cartilage. The ability of IL17 to synergise with other proinflammatory mediators to induce collagen release from cartilage, and what effect anti-inflammatory agents had on this process, was also assessed.

Methods: IL17 alone, or in combination with IL1, IL6, oncostatin M (OSM), or tumour necrosis factor alpha (TNFalpha), was added to bovine nasal cartilage explant cultures.

Amelioration of arthritis in two murine models using antibodies to oncostatin M.

Objective: Oncostatin M (OSM) is a member of the interleukin-6 cytokine family, with well-documented effects on cell growth and differentiation. OSM also has proinflammatory and cartilage degradative properties. The aim of this study was to investigate the significance of OSM in arthritis pathology using a neutralizing antibody in arthritis models.

Synergistic effects of glycoprotein 130 binding cytokines in combination with interleukin-1 on cartilage collagen breakdown.

Objective: To determine whether other glycoprotein 130 (gp130) binding cytokines can mimic the effects of oncostatin M (OSM) in acting synergistically with interleukin-1alpha (IL-1alpha) to induce cartilage collagen breakdown and collagenase expression, and to determine which receptors mediate these effects.

Methods: The release of collagen and proteoglycan was assessed in bovine and human cartilage explant cultures. Messenger RNA (mRNA) and protein production from immortalized human chondrocytes (T/C28a4) was analyzed by Northern blotting and specific enzyme-linked immunosorbent assays.

Type 1 IFN maintains the survival of anergic CD4+ T cells.

Anergic T cells have immunoregulatory activity and can survive for extended periods in vivo. It is unclear how anergic T cells escape from deletion, because both anergy and apoptosis can occur after TCR ligation. Stimulation of human CD4+ T cell clones reactive to influenza hemagglutinin peptides can occur in the absence of APCs when MHC class II-expressing, activated T cells present peptide to each other.

Discovery of novel ansamycins possessing potent inhibitory activity in a cell-based oncostatin M signalling assay.

We describe the isolation and characterisation of novel non-benzoquinone ansamycin metabolites related to geldanamycin from a culture of Streptomyces sp. S6699. The compounds possess potent inhibitory activity in a cell-based assay measuring inhibition of oncostatin M signalling in a reporter cell line utilising a secreted placental alkaline phosphatase (sPAP) readout.

The generation and characterization of antagonist RNA aptamers to human oncostatin M.

Oncostatin M (OSM) is a multifunctional member of the interleukin-6 cytokine family. OSM has been implicated as a powerful proinflammatory mediator and may represent a potentially important, novel therapeutic opportunity for treatment of established rheumatoid arthritis. To further investigate the role of OSM in inflammatory disorders, we have isolated a series of RNA aptamers that bind specifically to human OSM.

96-Well plate assays for measuring collagenase activity using (3)H-acetylated collagen.

We describe two alternative assays for measuring collagenolytic activity using (3)H-acetylated collagen. Both assays have been developed for the 96-well plate format and measure the amount of radiolabeled collagen fragments released into the supernatant from an insoluble (3)H-acetylated collagen fibril preparation. The first method separates digested solubilized fragments from the intact fibril by sedimentation of the undigested collagen by centrifugation.

A lack of evidence for down-modulation of CD3 zeta expression in colorectal carcinoma and pregnancy using multiple detection methods.

Loss of the T cell receptor-associated CD3 zeta chain has been proposed as a possible mechanism of the acquired immunosuppression in both tumour-bearing hosts, and in symptomatic patients with HIV infection. However, other reports suggest that the zeta-chain loss may in part be caused by protease activity of contaminating phagocytes ex vivo. Using flow cytometry and Western blot analysis on highly purified T cells, and ensuring adequate addition of protease inhibitors, we have studied the expression of CD3zeta on peripheral blood T cells from patients with colorectal carcinoma, and compared these with normal controls, and pregnant donors, as a further example of an immunocompromised state.

The role of oncostatin M in animal and human connective tissue collagen turnover and its localization within the rheumatoid joint.

Objective: To study the interaction of interleukin-1alpha (IL-1alpha) and oncostatin M (OSM) in promoting cartilage collagen destruction.

Methods: Bovine, porcine, and human cartilage and human chondrocytes were studied in culture. The levels of collagenase (matrix metalloproteinase 1 [MMP-1]) and tissue inhibitor of metalloproteinases 1 (TIMP-1) were measured by bioassay and enzyme-linked immunosorbent assay (ELISA).

Pairs of surface molecules involved in human IgE regulation: CD23-CD21 and CD40-CD40L.

At least two cell-derived signals have been shown to be necessary for the induction of immunoglobulin isotype switching in B-cells. The first signal is given by either of the soluble lymphokines, interleukin (IL)-4 or IL-13, which induce germline epsilon transcript expression, but this alone is insufficient to trigger secretion of immunoglobulin E (IgE). The second signal is provided by a physical interaction between B-cells and activated T-cells, basophils and mast cells, and it has been shown that the CD40/CD40 ligand (CD40L) pairing is crucial for mediating IgE synthesis.

IL-12 synergizes with IL-2 and other stimuli in inducing IL-10 production by human T cells.

IL-12, a potent inducer of IFN-gamma production by T cells and NK cells, has been recently reported to exacerbate an established Th2 response in vivo. However, the effect of IL-12 on Th2-lymphokine production remains unclear. Since IL-10 is a lymphokine associated with Th2 responses which decreases both IL-12-induced IFN-gamma production and IL-12 production by macrophages, we have analyzed here, in an APC-free system, the ability of IL-12 to modulate the production of human IL-10 by established Th0, Th1, and Th2 T cell clones (TCC), T cell lines, and purified peripheral blood T cells.

T-cell response to mycobacterial proteins: a comparative study of tuberculous and control immunoblots of Mycobacterium tuberculosis and M. bovis BCG.

Objective: To evaluate and compare the lymphoproliferative response of human peripheral blood mononuclear cells (PBMC) to fractionated soluble extracts of Mycobacterium tuberculosis H37Rv (MTSE) and M. bovis bacille Calmette-Guérin (BCG) (MBSE), and thereby determine responses that correlate to infection, and to contrast antibody and T-cell responses.

Design: Membrane blots of SDS-PAGE fractionated M.

Thiols decrease human interleukin (IL) 4 production and IL-4-induced immunoglobulin synthesis.

N-Acetyl-L-cysteine (NAC) is an antioxidant precursor of intracellular glutathione (GSH), usually given in human as a mucolytic agent. In vitro, NAC and GSH have been shown to act on T cells by increasing interleukin (IL) 2 production, synthesis and turnover of IL-2 receptors, proliferation, cytotoxic properties, and resistance to apoptosis. We report here that NAC and GSH decrease in a dose-dependent manner human IL-4 production by stimulated peripheral blood T cells and by T helper (Th) 0- and Th2-like T cell clones.

Interleukin-12 increases interleukin-4 production by established human Th0 and Th2-like T cell clones.

Interleukin (IL)-12 is a potent inducer of cell-mediated immunity: it favors the generation of interferon (IFN)-gamma-producing T cells, increases IFN-gamma production by T cells and natural killer cells and prevents the generation of a Th2 response in murine in vivo models. Nevertheless, the effects of IL-12 on an established Th2 response remain poorly documented. In the present paper, we analyzed the effect of IL-12 on the profile of lymphokines produced by established IL-4-producing Th0 and Th2-like human T cell clones (TCC) and by polyclonal T cells.

CD23 regulates monocyte activation through a novel interaction with the adhesion molecules CD11b-CD18 and CD11c-CD18.

CD23 is expressed on a variety of haemopoietic cells and displays pleiotropic activities in vitro. We report that in addition to CD21 and IgE, CD23 interacts specifically with the CD11b and CD11c, the alpha chains of the beta 2 integrin adhesion molecule complexes CD11b-CD18 and CD11c-CD18, on monocytes. Full-length recombinant CD23 incorporated into fluorescent liposomes was shown to bind to COS cells transfected with cDNA encoding either CD11b-CD18 or CD11c-CD18 but not with CD11a-CD18.

Regulation of IgE synthesis by CD23/CD21 interaction.

At least two cell-derived signals have been shown to be necessary for the induction of immunoglobulin isotype switching in B cells. The first signal is given by either of the soluble lymphokines interleukin (IL)-4 or IL-13 which induce germline epsilon transcript expression, but alone is insufficient to trigger secretion of IgE. The second signal is provided by a physical interaction between B cells and activated T cells, basophils and mast cells, and it has been shown that the CD40/CD40L pairing is crucial for mediating IgE synthesis.

IL-8-induced signal transduction in T lymphocytes involves receptor-mediated activation of phospholipases C and D.

We have characterized the IL-8-induced signal transduction processes in T lymphocytes. A basal level of IL-8 receptor expression was shown on mixed PBL, as identified by using phycoerythrin (PE)-coupled IL-8, and this expression was increased following IL-2 stimulation. Scatchard analysis of T cells revealed competitive binding of IL-8 with a Kd of 0.

CD28 functions as an adhesion molecule and is involved in the regulation of human IgE synthesis.

Activated T cells induce IgE switching in B cells via a combination of lymphokines and direct T:B cell contact. As CD28-deficient mice have reduced basal levels of IgG1 and IgG2a and diminished Ig class switching, we investigated whether the CD28/B7.1 (CD80) ligand pairing might also be involved in human IgE regulation.