ABSTRACT
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. To our knowledge this is the first report showing a close pattern of expression between mRNA and protein for IL-1beta and IL-6, but not for TNF-alpha, in these two models of RA. The kinetics of expression for these biomarkers suggested that the optimal sampling time-points to study the effect of compounds on both inflammation and cytokine levels were day 4 postonset in CIA and day 3 after i.v challenge in SCW-induced arthritis. Prednisolone reduced joint swelling through a mechanism associated with a reduction in IL-1beta and IL-6 protein and mRNA expression levels. At the investigated time points, protein levels for TNF-alpha in arthritic joints were lower than the lower limit of detection of the ELISA, whereas mRNA levels for this cytokine were reliably detected. These observations suggest that RT-PCR TaqMan is a sensitive technique that can be successfully applied to the quantification of mRNA levels in rodent joints from experimental arthritis models providing insights into mechanisms of action of novel anti-inflammatory drugs.
Subject(s)
Anti-Inflammatory Agents/therapeutic use , Arthritis, Experimental/immunology , Cytokines/analysis , Prednisolone/therapeutic use , Proteins/analysis , RNA, Messenger/analysis , Animals , Arthritis, Experimental/drug therapy , Base Sequence , Biomarkers/analysis , Enzyme-Linked Immunosorbent Assay/methods , Interleukin-1/analysis , Interleukin-6/analysis , Joints/immunology , Male , Mice , Mice, Inbred DBA , Rats , Rats, Inbred Lew , Time Factors , Tumor Necrosis Factor-alpha/analysisABSTRACT
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. Proteoglycan and collagen release were determined. Collagenolytic activity was determined by bioassay. Chondroprotective effects of IL4, IL13, transforming growth factor beta1 (TGFbeta1) and insulin-like growth factor-1 (IGF1) were assessed by inclusion in the explant cultures. RESULTS: IL17 alone stimulated a dose dependent release of proteoglycan and type II collagen from bovine nasal cartilage explants. Suboptimal doses of IL17 synergised potently with TNFalpha, IL1, OSM, and IL6 to promote collagen degradation. This collagen release was completely inhibited by tissue inhibitor of metalloproteinase-1 and BB-94 (a synthetic metalloproteinase inhibitor), and was significantly reduced by IL4, IL13, TGFbeta1, and IGF1. In IL17 treated chondrocytes, mRNA expression for matrix metalloproteinase (MMP)-1, MMP-3, and MMP-13 was detected. Moreover, a synergistic induction of these MMPs was seen when IL17 was combined with other proinflammatory cytokines. CONCLUSIONS: IL17 can, alone and synergistically in combination with other proinflammatory cytokines, promote chondrocyte mediated MMP dependent type II collagen release from cartilage. Because levels of all these proinflammatory cytokines are raised in rheumatoid synovial fluids, this study suggests that IL17 may act as a potent upstream mediator of cartilage collagen breakdown in inflammatory joint diseases.
Subject(s)
Cartilage/drug effects , Collagen/drug effects , Interleukin-17/pharmacology , Animals , Cartilage/metabolism , Cattle , Collagen/metabolism , Collagen Type II/drug effects , Collagen Type II/metabolism , Drug Combinations , Drug Synergism , Interleukin-1/pharmacology , Interleukin-6/pharmacology , Metalloendopeptidases/pharmacology , Oncostatin M , Peptides/pharmacology , ProteoglycansABSTRACT
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. METHODS: Collagen-induced arthritis (CIA) was established in male DBA/1 mice. Reverse transcriptase-polymerase chain reaction was used to detect OSM messenger RNA (mRNA) message levels in arthritic joints. Neutralizing anti-OSM antibody or control immunoglobulin was administered on days 1 and 3 after disease onset. Animals were assessed for clinical arthritis for 2 weeks, followed by a histologic analysis of paws. Pristane-induced arthritis (PIA) was produced in male CBA mice dosed with anti-OSM or control immunoglobulin immediately before disease onset. Mice with PIA were assessed for clinical arthritis over a period of 100 days. RESULTS: Levels of mRNA for OSM, but not GAPDH, were elevated in arthritic joints of mice with CIA compared with those of normal controls. Mice with CIA treated with anti-OSM antibody showed significant amelioration of both the clinical severity (P < 0.01) and the number of affected paws (P < 0.01) compared with control animals. Histologic analysis confirmed these clinical findings, revealing a marked reduction in cellular infiltration of synovium and cartilage damage. In the PIA model, the incidence of arthritis was 65% in the control group compared with 0% in the anti-OSM-treated animals. CONCLUSION: These results demonstrate a key role for endogenously produced OSM as a potent mediator of joint pathology, and suggest that OSM might be a potentially important, novel therapeutic target for treatment of established rheumatoid arthritis.
Subject(s)
Antibodies/therapeutic use , Arthritis, Experimental/drug therapy , Bacterial Proteins , Peptides/metabolism , Animals , Arthritis, Experimental/chemically induced , Arthritis, Experimental/pathology , Chaperonin 60 , Chaperonins/pharmacology , Collagen/immunology , Collagen/pharmacology , DNA Primers/chemistry , Disease Models, Animal , Edema/drug therapy , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Glyceraldehyde-3-Phosphate Dehydrogenases/immunology , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Immunoglobulin G/administration & dosage , Joints/chemistry , Joints/drug effects , Joints/metabolism , Joints/pathology , Male , Mice , Mice, Inbred CBA , Mice, Inbred DBA , Oncostatin M , Peptides/genetics , Peptides/immunology , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Terpenes/immunology , Terpenes/pharmacologyABSTRACT
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. Collagenase activity was measured by bioassay. Cell surface receptors were detected by flow cytometry. RESULTS: OSM in combination with IL-1alpha caused a rapid synergistic induction of matrix metalloproteinase 1 mRNA, which was sustained over a 72-hour period. Flow cytometric analyses detected both the OSM-specific receptor and the gp130 receptor at the chondrocyte cell surface, but failed to detect the leukemia inhibitory factor receptor (LIFR). Cartilage degradation assays revealed that, of the gp130 binding cytokines, only OSM and IL-6, in the presence of its soluble receptor (sIL-6R), were able to act synergistically with IL-1alpha to promote collagen release. CONCLUSION: This study demonstrates that IL-6 can mimic OSM in synergizing with IL-1alpha to induce chondrocyte-mediated cartilage collagen breakdown and collagenase production. In order to have this effect, IL-6 requires the presence of its soluble receptor. The apparent absence of LIFR explains why other gp130 binding cytokines do not act in synergy with IL-1alpha. Since OSM, IL-6, and sIL-6R levels have all been shown to be elevated in the rheumatoid joint, our findings suggest that these cytokines may be key mediators of cartilage collagen catabolism in the inflammatory arthritides.
Subject(s)
Antigens, CD/metabolism , Chondrocytes/drug effects , Collagen/metabolism , Interleukin-1/pharmacology , Interleukin-6/pharmacology , Membrane Glycoproteins/metabolism , Animals , Cartilage, Articular/cytology , Cattle , Cell Line, Transformed , Chondrocytes/cytology , Chondrocytes/enzymology , Ciliary Neurotrophic Factor/metabolism , Ciliary Neurotrophic Factor/pharmacology , Cytokine Receptor gp130 , Cytokines/metabolism , Cytokines/pharmacology , Drug Synergism , Gene Expression Regulation, Enzymologic/drug effects , Growth Inhibitors/metabolism , Growth Inhibitors/pharmacology , Humans , Interleukin-1/metabolism , Interleukin-11/metabolism , Interleukin-11/pharmacology , Interleukin-6/metabolism , Leukemia Inhibitory Factor , Lymphokines/metabolism , Lymphokines/pharmacology , Matrix Metalloproteinase 1/genetics , Matrix Metalloproteinase 1/metabolism , Oncostatin M , Peptides/metabolism , Peptides/pharmacology , RNA, Messenger/analysis , Receptors, Interleukin-6/metabolism , Tissue Inhibitor of Metalloproteinase-1/genetics , Tissue Inhibitor of Metalloproteinase-1/metabolism , Tissue Inhibitor of Metalloproteinase-2/genetics , Tissue Inhibitor of Metalloproteinase-2/metabolismABSTRACT
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. This T:T peptide presentation can induce CD95-mediated apoptosis, while the cells that do not die are anergic. We found that the death after peptide or anti-CD3 treatment of a panel of CD4+ T cell clones is blocked by IFN-beta secreted by fibroblasts and also by IFN-alpha. This increases cell recovery after stimulation, which is not due to T cell proliferation. This mechanism for apoptosis inhibition rapidly stops protein kinase C-delta translocation from the cytoplasm to the nucleus, which is an early event in the death process. A central observation was that CD4+ T cells that are rescued from apoptosis after T:T presentation of peptide by IFN-alphabeta remain profoundly anergic to rechallenge with Ag-pulsed APCs. However, anergized cells retain the ability to respond to IL-2, showing that they are nonresponsive but functional. The prevention of peptide-induced apoptosis in activated T cells by IFN-alphabeta is a novel mechanism that may enable the survival and maintenance of anergic T cell populations after TCR engagement. This has important implications for the persistence of anergic T cells with the potential for immunoregulatory function in vivo.
Subject(s)
CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , Cell Survival/immunology , Clonal Anergy/immunology , Interferon Type I/physiology , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/immunology , Antigen-Presenting Cells/immunology , Apoptosis/immunology , Clone Cells , Coculture Techniques , Culture Media, Conditioned/chemistry , Culture Media, Conditioned/pharmacology , Fibroblasts/chemistry , Fibroblasts/immunology , Fibroblasts/metabolism , Humans , Interferon-alpha/physiology , Interferon-beta/physiology , Interleukin-2/physiology , Peptides/immunology , Peptides/pharmacologyABSTRACT
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. In this paper we report the isolation and structure elucidation of the compounds and describe some of their biological properties.
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Peptides/metabolism , Alkaline Phosphatase/drug effects , Alkaline Phosphatase/genetics , Alkaline Phosphatase/metabolism , Anti-Bacterial Agents/metabolism , Benzoquinones , Cell Line , Cell Survival/drug effects , Drug Evaluation, Preclinical/methods , Humans , Inhibitory Concentration 50 , Interleukin-6/metabolism , Interleukin-6/pharmacology , Lactams, Macrocyclic , Lung/cytology , Lung/drug effects , Oncostatin M , Protein Biosynthesis , Proteins/drug effects , Quinones/chemistry , Quinones/metabolism , Quinones/pharmacology , Recombinant Proteins/drug effects , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Rifabutin/chemistry , Rifabutin/pharmacology , Signal Transduction , Streptomyces/metabolismABSTRACT
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. The highest affinity aptamer, designated ADR58, has been characterized in a series of in vitro and cell based assays. ADR58 has an affinity of 7 nm for human OSM, and it can antagonize OSM binding to the gp130 receptor and specifically antagonize OSM mediated signaling. The aptamer has been truncated in length to 33 bases, all pyrimidine positions are substituted with 2' fluorine, and 14 of 18 purine positions have been substituted with 2' O-methyl to increase stability toward nucleases. This truncated, modified form of ADR58 retains complete affinity and functional activity for OSM. This aptamer may be used as a tool to further investigate the role of OSM in inflammatory disorders and may also have role as a therapeutic agent.
Subject(s)
Inflammation Mediators/antagonists & inhibitors , Peptides/antagonists & inhibitors , RNA/pharmacology , Animals , Arthritis, Rheumatoid/etiology , Arthritis, Rheumatoid/therapy , Base Sequence , Humans , Mice , Molecular Sequence Data , Oncostatin M , Peptides/physiologyABSTRACT
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. The second method achieves this separation by filtration of the supernatant through the membrane of a 96-well filtration plate which retains the undigested collagen fibril. Both methods give linear dose- and time-dependent responses of collagenase activity > or = 70% of total collagen lysis. In addition, both assays can be simply modified to measure tissue inhibitors of metalloproteinases (TIMPs) inhibitory activity, which is also linear between 20 and 75% of total collagen lysis with the amount of TIMP added.
Subject(s)
Collagen/metabolism , Collagenases/metabolism , Acetylation , Animals , Matrix Metalloproteinase Inhibitors , Rabbits , TritiumABSTRACT
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. Immunohistochemistry was performed on tumour sections from patients with colorectal carcinoma to measure CD3zeta expression in tumour infiltrating T cells, and compared with normal mucosa and tonsil. Using these three approaches, our data provide no evidence for downregulation of CD3zeta chain expression either in colorectal carcinoma or pregnancy and suggest that this explanation is unlikely to fully account for the reduced T cell function associated with these conditions in all patients.
Subject(s)
Adenocarcinoma/metabolism , CD3 Complex , Colorectal Neoplasms/metabolism , Down-Regulation/immunology , Membrane Proteins/biosynthesis , Pregnancy/immunology , Receptors, Antigen, T-Cell/biosynthesis , Adenocarcinoma/chemistry , Adenocarcinoma/immunology , Adult , Aged , Aged, 80 and over , Blotting, Western , Colorectal Neoplasms/chemistry , Colorectal Neoplasms/immunology , Female , Flow Cytometry , Humans , Immunohistochemistry , Male , Membrane Proteins/immunology , Middle Aged , Receptors, Antigen, T-Cell/chemistry , Receptors, Antigen, T-Cell/immunologyABSTRACT
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). The levels of OSM in rheumatoid synovial fluid were measured by ELISA. RESULTS: When combined with OSM, IL-1alpha, IL-1beta, and tumor necrosis factor alpha released proteoglycan and collagen from cartilage. OSM was the only member of the IL-6 family to have this effect. Human tendon also responded to IL-1alpha and OSM. OSM increased the production of MMP-1 and TIMP-1 but when combined with IL-1alpha, synergistically promoted MMP-1 production in human chondrocytes and synovial fibroblasts. High levels of OSM were found in human rheumatoid synovial fluids, and confocal microscopy showed that OSM was produced by macrophages in rheumatoid synovial tissue. CONCLUSION: These results highlight an important new mechanism by which there is irreversible loss of collagen from cartilage.
Subject(s)
Arthritis, Rheumatoid/metabolism , Collagen/metabolism , Connective Tissue/chemistry , Growth Inhibitors/physiology , Peptides/physiology , Animals , Blotting, Northern , Blotting, Western , Cartilage, Articular/drug effects , Cartilage, Articular/enzymology , Cartilage, Articular/metabolism , Cattle , Chondrocytes/drug effects , Collagenases/metabolism , Cytokines/metabolism , Humans , Interleukin-1/pharmacology , Interleukin-6/pharmacology , Knee Joint/chemistry , Microscopy, Confocal , Oncostatin M , Osteoarthritis/metabolism , Phenotype , Swine , Synovial Fluid/chemistry , Synovial Membrane/chemistry , Synovial Membrane/metabolism , Tendons/drug effectsABSTRACT
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. In hyper-immunoglobulin M1 (HIGM1) syndrome, which is characterized by greatly decreased levels of immunoglobulin G, A and E (IgG, IgA and IgE), there are mutations in the CD40L resulting in a completely non-functional extracellular domain. The CD40L is, therefore, playing a central role in immunoglobulin isotype switching. Amongst the numerous pairs of surface adhesion molecules, the CD23-CD21 pair seems to play a key role in the generation of IgE. The CD23 molecule is positively and negatively regulated by factors which increase or decrease IgE production, respectively. Antibodies to CD23 have been shown to inhibit IL-4-induced human IgE production in vitro and to inhibit antigen-specific IgE responses in a rat model, in an isotype selective manner. CD23 interacts with CD21 on B-cells, preferentially driving IgE production. CD23 recognizes two main epitopes on the CD21 molecule. One region consists of short consensus repeat (SCR) sequences 1-2 and the other of SCR 5-8. In the latter region, Asn 370 and 295 are critical in the interaction with the lectin CD23. Therefore, a restricted number of cytokines and surface molecules seems to selectively regulate human immunoglobulin E synthesis.
Subject(s)
Antigens, Surface/immunology , CD40 Antigens/immunology , Hypergammaglobulinemia/immunology , Immunoglobulin Class Switching/immunology , Immunoglobulin E/biosynthesis , Immunoglobulin M/biosynthesis , Receptors, Complement 3d/immunology , Receptors, IgE/immunology , Animals , Antigens, Surface/physiology , B-Lymphocytes/immunology , CD40 Antigens/genetics , CD40 Antigens/physiology , Eosinophils/immunology , Humans , Immunoglobulin A/biosynthesis , Immunoglobulin G/biosynthesis , Interleukin-13/immunology , Interleukin-4/immunology , Mast Cells/immunology , Rats , Receptors, Complement 3d/physiology , Receptors, IgE/genetics , Receptors, IgE/physiology , Signal Transduction , SyndromeABSTRACT
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. IL-12 synergized with anti-CD3 mAb, Con A, or IL-2 in inducing IL-10 production by Th0, Th1, and Th2 TCC and by T cell lines. This effect was dose dependent (from 0.1 to 50 U/ml) and associated with an increase of IL-10 mRNA transcription. As previously reported, IL-12 also enhanced IFN-gamma production by stimulated Th1 and Th0 TCC and, to a lesser extent, IL-4 production by stimulated Th0 and Th2 TCC. These observations were extended to peripheral blood T cells stimulated in the presence of exogenous IL-2. Moreover, using neutralizing anti-IL-2 Ab, we report that endogenous IL-2 produced by stimulated Th0 TCC could in part contribute to the effect of IL-12 on IL-10 and IL-4 production. In conclusion, IL-12 synergizes with IL-2 and other stimuli in inducing IL-4 and IL-10 production by T cells. This property may help to explain why IL-12 does not efficiently down-regulate an established Th2 response.
Subject(s)
Interleukin-10/biosynthesis , Interleukin-12/pharmacology , Interleukin-2/pharmacology , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , Adjuvants, Immunologic/pharmacology , Antibodies, Monoclonal/pharmacology , Base Sequence , CD3 Complex/immunology , Cell Line , Cells, Cultured , Clone Cells/metabolism , Drug Synergism , Humans , Interleukin-12/physiology , Interleukin-4/biosynthesis , Interphase/immunology , Lymphocyte Activation/drug effects , Molecular Sequence DataABSTRACT
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. tuberculosis H37Rv and M. bovis BCG were employed for antibody immunoblotting and T-cell proliferative responses using sera and PBMC from seven tuberculous and seven BCG vaccinated control subjects. RESULTS: The profiles of responses contrasted rather interestingly, with antibody and T-cells responding more to higher and lower molecular weight fractions respectively. T-cells responding to antigens in the 59-88 kDa region discriminated between tuberculous and BCG vaccinated controls (P < 0.05) even though the differences were more toward the 70-75 kDa fractions within the region in question. Responses to smaller molecular weight fractions of both MTSE and MBSE were high in direct contrast to antibody responses. Additionally, responses to MBSE in these regions were generally higher than for MTSE in vaccinated controls. The reverse was the case with tuberculous subjects where responses to MTSE were generally higher, though not sufficiently significant in enough of the tuberculous subjects to be considered discriminatory. CONCLUSION: T-cell proliferative responses to mycobacterial antigens in the 59-88 kDa region, and particularly antigens in the 70-75 kDa region, can be an indication of infection with M. tuberculosis, as well as the basis for discriminating between active disease and vaccination with BCG.
Subject(s)
Antigens, Bacterial/immunology , Mycobacterium bovis/immunology , Mycobacterium tuberculosis/immunology , T-Lymphocytes/immunology , Tuberculosis/immunology , Antigens, Bacterial/chemistry , Blotting, Western , Cell Culture Techniques , Cell Division/immunology , Clone Cells/immunology , Humans , Lymphocyte Activation , Molecular WeightABSTRACT
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. This effect was associated with a decrease in IL-4 messenger RNA transcription. In contrast, NAC and GSH had no effect on interferon gamma and increased IL-2 production and T cell proliferation. A functional consequence was the capacity of NAC and GSH to selectively decrease in a dose-dependent manner IL-4-induced immunoglobulin (Ig) E and IgG4 production by human peripheral blood mononuclear cells. Interestingly, NAC and GSH also acted directly on purified tonsillar B cells by decreasing the mature epsilon messenger RNA, hence decreasing IgE production. In contrast, IgA and IgM production were not affected. At the same time, B cell proliferation was increased in a dose-dependent manner. Not all antioxidants tested but only SH-bearing molecules mimicked these properties. Finally, when given orally to mice, NAC decreased both IgE and IgG1 antibody responses to ovalbumin. These results demonstrate that NAC, GSH, and other thiols may control the production of both the Th2-derived cytokine IL-4 and IL-4-induced Ig in vitro and in vivo.
Subject(s)
Acetylcysteine/pharmacology , Antioxidants/pharmacology , Immunoglobulins/biosynthesis , Interleukin-4/biosynthesis , Sulfhydryl Compounds/pharmacology , T-Lymphocyte Subsets/immunology , Animals , Antibody Formation/drug effects , B-Lymphocytes/physiology , CD40 Antigens/genetics , Cells, Cultured , Gene Expression/drug effects , Genes, Immunoglobulin , Humans , Interleukin-4/genetics , Lymphocyte Activation/drug effects , Mice , Mice, Inbred C57BL , Palatine Tonsil/cytology , RNA, Messenger/genetics , Receptors, Complement 3d/geneticsABSTRACT
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. We found that IL-12 (i) enhances, as previously reported, IFN-gamma production by Th0 TCC and, to a smaller extent, by Th2-like TCC, (ii) increases the proliferation of Th0 and Th2-like TCC and (iii) unexpectedly, synergizes with T cell receptor-associated or nonspecific stimuli in increasing IL-4 production by these TCC. Thus, IL-12 potentiates the production of IFN-gamma and also of IL-4 by established IL-4 producing TCC. Although IL-12 has been widely reported to induce a Th1 response and to prevent the development of a Th2 response in vivo, IL-12 may on the contrary potentiate an established Th2 response in humans.
Subject(s)
Interleukin-12/physiology , Interleukin-4/biosynthesis , T-Lymphocytes, Helper-Inducer/metabolism , Th2 Cells/metabolism , CD4-Positive T-Lymphocytes/chemistry , Clone Cells , Humans , Interferon-gamma/biosynthesis , Lymphocyte Activation/immunology , Tumor Cells, CulturedABSTRACT
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. The interaction was specifically inhibited by anti-CD11b or anti-CD11c, respectively, and by anti-CD23 MAbs. The functional significance of this ligand pairing was demonstrated by triggering CD11b and CD11c on monocytes with either recombinant CD23 or anti-CD11b and anti-CD11c MAbs to cause a marked increase in nitrite-oxidative products and pro-inflammatory cytokines (IL-1 beta, IL-6, and TNF alpha). These CD23-mediated activities were decreased by Fab fragments of MAbs to CD11b, CD11c, and CD23. These results demonstrate that CD11b and CD11c are receptors for CD23 and that this novel ligand pairing regulates important activities of monocytes.
Subject(s)
CD11 Antigens/metabolism , CD18 Antigens/metabolism , Monocytes/metabolism , Receptors, IgE/metabolism , Cell Adhesion Molecules/metabolism , Cells, Cultured , Cytokines/biosynthesis , Humans , Hydrogen Peroxide/metabolism , Liposomes , Signal TransductionSubject(s)
CD4-Positive T-Lymphocytes/drug effects , Colon/virology , Cytokines/pharmacology , HIV Envelope Protein gp120/drug effects , HIV-1/pathogenicity , Intestinal Mucosa/virology , Colon/immunology , HIV Envelope Protein gp120/metabolism , HIV-1/drug effects , Humans , Intestinal Mucosa/drug effects , Intestinal Mucosa/immunology , Protein Binding/drug effects , Protein Binding/immunologyABSTRACT
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. In HIGM1 syndrome, which is characterized by greatly decreased levels of IgG, IgA and IgE, there are mutations in CD40L resulting in a completely non-functional extracellular domain. CD40L is therefore playing a central role in Ig switching. Amongst the numerous pairs of surface adhesion molecules, the CD23/CD21 pair seems to play a key role in the generation of IgE. The CD23 molecule is positively and negatively regulated by factors which increase or decrease IgE production, respectively. Antibodies to CD23 have been shown to inhibit IL-4-induced human IgE production in vitro and to inhibit antigen-specific IgE responses in a rat model, in an isotype-selective manner. CD23 interacts with CD21 on B cells, preferentially driving IgE production. CD23 recognises two main epitopes on the CD21 molecule. One region consists of short consensus repeat sequences (SCRs) 1-2 and the other of SCRs 5-8. In the latter region ASn370 and Asn295 are critical in the interaction with the lectin CD23. Therefore, a restricted number of cytokines and surface molecules seems to selectively regulate human IgE synthesis.
Subject(s)
Immunoglobulin Class Switching/physiology , Immunoglobulin E/biosynthesis , Receptors, Complement 3d/physiology , Receptors, IgE/physiology , Animals , Antigens, CD/physiology , Antigens, Differentiation, B-Lymphocyte/physiology , B-Lymphocytes/immunology , CD40 Antigens , CD40 Ligand , Cell Adhesion , Consensus Sequence , Herpesvirus 4, Human/physiology , Humans , Interleukin-13/pharmacology , Interleukin-4/pharmacology , Lymphocyte Cooperation , Membrane Glycoproteins/physiology , Mutagenesis, Site-Directed , Receptors, Complement 3d/genetics , Signal Transduction , T-Lymphocytes/immunology , Viral Proteins/pharmacologyABSTRACT
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 0.55 nM, with approximately 1200 receptors per cell, on freshly isolated T cells. After 24 h in culture following purification, reverse transcriptase PCR (RT-PCR) analyses show the mRNA for only the type B IL-8R on these cultured T lymphocytes and the cell line MOLT-4. Stimulation of T lymphocytes or T cell clones with IL-8 led to generation of inositol trisphosphate and calcium flux. In addition, when T cells were prelabeled with [3H]oleic acid, IL-8 caused a long lasting, time- and dose-related increase in [3H]phosphatidylethanol (PtE), indicating activation of phospholipase D (PLD). By contrast, this IL-8-dependent PLD activity was undetectable in IL-8-stimulated neutrophils. PLD activation appeared to be downstream of protein kinase C, because several inhibitors abrogated the increase in [3H]PtE, whereas guanosine-5'-O-(3-thiotriphosphate (GTP(gamma)S) and inositol trisphosphorothioate (IP3S3) both increased the generation of [3H]PtE. Together, these results demonstrate that the IL-8RB receptor is sufficient to mediate phospholipase C (PLC) and PLD activation in T lymphocytes, but not in neutrophils, and indicate an important difference in receptor usage and signal transduction pathways between IL-8-stimulated lymphocytes and neutrophils.