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D Lappin

Publications and source records attributed to D Lappin.

At least 19 recordsLinked to original sources
PubMed1994

Expression of the components and regulatory proteins of the classical pathway of complement in normal and diseased synovium.

We studied the synthesis of the classical pathway complement components in synovial membrane. Ribonucleic acid was extracted from the synovial membranes of patients with rheumatoid arthritis (RA) or osteoarthritis (OA), as well as from normal synovial membrane. Northern blot and dot blot analysis showed that the mRNAs for all classical pathway complement components (C1qA chain, C1qB chain, C1qC chain, C1r, C1s, C4 and C2) and the fluid-phase regulatory components (C1-inhibitor, C4-bp and factor I) were present in all three types of synovial membrane. Thus, all the components of the classical pathway were expressed in normal and diseased synovium. In an attempt to determine which components were synthesised by each cell type, monocytes (mononuclear phagocytes), human umbilical vein endothelial cells (HUVEC), synovial membrane fibroblasts (from normal, OA and RA synovial membrane) and peripheral blood lymphocytes were cultured in vitro and secretion rates of individual components were measured and total cellular RNA was analysed by Northern blotting. Monocytes secreted C1q, C1r, C1s, C4, C2, C1-inhibitor and C4-bp but not factor I. Fibroblasts secreted C1r, C1s, C2, C3, C1-inhibitor and factor I but not C1q, C4 or C4-bp. HUVEC secreted C1s, C2, C1-inhibitor and factor I but not C1q, C1r, C4 or C4-bp. Lymphocytes did not secrete any of these components. In three instances mRNA was detected in the absence of secreted protein: mRNAs for the C1qA and C1qC chains were detected in HUVEC, whereas the mRNA for the C1qB chain was not, and C4 mRNA was detected in both fibroblasts and HUVEC.(ABSTRACT TRUNCATED AT 250 WORDS)

Arthritis, Rheumatoid↗
PubMed1993

Regulation of the synthesis of C1 subcomponents and C1-inhibitor.

We have investigated the synthesis of C1q, C1r, C1s and C1-inhibitor in HepG2 cells, human umbilical vein endothelial cells (HUVEC), fibroblasts (skin and synovial membrane), chondrocytes and monocytes. C1q was only synthesised by monocytes, although the mRNAs for the C1qA and C1qC chains were expressed in HUVEC. C1r, C1s and C1-inhibitor were synthesised by all cell types. The secretion rates of C1r and C1s were approximately equimolar in fibroblasts and chondrocytes whereas the secretion rate for C1s exceeded that for C1r in the other cell types. Molar ratios of C1s to C1r were approximately 2:1 for HepG2 cells, 5:1 for monocytes and 10:1 for HUVEC. Stimulation with interferon-gamma resulted in increased expression of all four proteins. The C1s:C1r ratio did not alter in chondrocytes or fibroblasts, but approached unity in HepG2, monocytes and HUVEC, due to relatively greater stimulation of C1r gene expression.

Carcinoma, Hepatocellular↗
PubMed1989

Regulation of C1-inhibitor synthesis by interferons and other agents.

C1-inhibitor (C1-inh) is synthesised and secreted by at least four cell types: hepatocytes, mononuclear phagocytes, fibroblasts and umbilical vein endothelial cells. The production of this protein by monocytes/macrophages and Hep G2 cells has been studied in great detail. Environmental factors alter C1-inh synthesis by these cells. A number of agents which inhibit monocyte C1-inh production (such as histamine, PGE2 C5a des arg and serum treated immune complexes) bind to membrane receptors, activate adenylate cyclase, elevate intracellular cAMP and activate cAMP-dependent protein kinase. Elevation of monocyte cAMP levels is associated with decreased C1-inh secretion by these cells and reduced C1-inh mRNA levels. These changes can be seen within 8 hours of exposure. Stimulation of monocyte C1-inh synthesis occurs after the addition of agents which induce the formation of sodium ion and calcium ion channels, activate the phosphatidyl inositol cycle and activate protein kinase C (immune-complexes, carbamylcholine and phenylephrine). Agents which act directly on protein kinase C (phorbol myristate acetate) also stimulate C1-inh synthesis. Amongst the most potent stimulators of monocyte and Hep G2 C1-inh synthesis are the interferons (Ifns) Ifn alpha, Ifn beta and Ifn gamma. These are known to bind to specific receptors on cells (Ifn alpha and beta binding to Type I Ifn receptors and Ifn-gamma binding to type II Ifn receptors). At least two mechanisms by which Ifn receptor-ligand interaction elicit their effects exist. These are: 1) binding of an activated receptor transducer/regulatory component to specific DNA sequences on Ifn sensitive genes; 2) the activation of protein kinase C and binding of its regulatory components to specific DNA sequences. Ifn alpha, beta and gamma cause a dose related increase in monocyte and Hep G2 cell C1-inh mRNA abundance and protein synthesis. Ifn-gamma is the most potent of the interferons on monocyte C1-inh synthesis. Ifn alpha and beta being less effective but equipotent. These cytokines elicit their maximum effect on monocyte C1-inh synthesis after 1-2 hours treatment. This rapid stimulation of monocyte C1-inh synthesis suggests that this increases the transcription of the C1-inh gene. After removal of Ifns from monocytes the elevated C1-inh mRNA levels subside towards control levels of expression in Ifn alpha and beta-treated cells but remain elevated in Ifn-gamma-treated monocytes. This binding suggests that Ifn-gamma alters the stability of C1-inh mRNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Carcinoma, Hepatocellular↗
PubMed1989

Modulation of monocyte complement synthesis by lymphocytes and lymphocyte-conditioned media.

Culture supernatants from mitogen- and antigen-stimulated human peripheral blood lymphocytes (PBL), stimulated synthesis of the second complement component (C2) by human monocytes, but not as effectively as the stimulated PBL themselves, which adhered to the monocytes and caused marked spreading. In contrast to PBL, lymphocytes isolated from the synovial membranes (SML) of patients with rheumatoid arthritis and their culture supernatants were able to stimulate C2 synthesis without exposure to mitogens or antigens. Depletion of B and T populations showed that T cells were responsible for stimulation of C2 synthesis. Further studies of synthesis rates of C2, C3 factor B (B), C1 inhibitor, and properdin (P) were undertaken, and it was found that lymphocytes and their supernatants increased synthesis of C2, B and C1 inhibitor, and reduced synthesis of C3 and P. This profile of activity was identical to that produced by the addition of recombinant gamma-interferon (rIFN-gamma) to the cultures. Furthermore the addition of a monoclonal antibody to rIFN-gamma to cultures abrogated the effects of rIFN-gamma, and almost completely reversed the effects of lymphocytes and their supernatants. Thus it appears that gamma-interferon is the lymphocyte product which is responsible for the modulation of monocyte complement synthesis. The results of studies with synovial membrane lymphocytes raise the possibility that this process occurs in vivo. Monocyte C2 had a higher specific functional activity (SpFA) than serum C2 isolated from serum or C2 produced by HepG2 cells. Monocyte C2 formed a C3 convertase which had a longer half-life than that found with both serum C2 or HepG2 C2. Thus monocyte C2 behaves like oxidized C2. Monocytes exposed to rIFN-gamma, lymphocytes or lymphocyte-conditioned medium (LCM) produced C2 which had an even higher SpFA. Although antibody to IFN-gamma prevented any increase in C2 synthesis in monocyte cultures containing lymphocytes or LCM, C2 SpFA was still increased. Thus a second lymphocyte product is responsible for this 'oxidation' effect. This production of 'oxidized' C2 by monocytes and further 'oxidation' by the action of either lymphocytes or gamma-interferon might play a significant role in the perpetuation of complement activation at sites of inflammation.

Cells, Cultured↗
PubMed1989

Complement biosynthesis in human synovial tissue.

Molecular biological and immunochemical techniques have been used to study the synthesis of complement components by synovial tissue from patients with rheumatoid arthritis or osteoarthritis and by normal synovial tissue from a patient undergoing patellectomy. Using Northern and dot-blot analyses, mRNAs coding for C1-inhibitor, C2, C3, C4 and factor B have been detected, but not for C5. Quantitative analyses of the data have not shown any significant differences in the steady state levels of any of the mRNAs in synovium from rheumatoid arthritis and osteoarthritis patients. When synovial membrane fragments from rheumatoid arthritis, osteoarthritis patients or normal synovium were cultured in vitro, synthesis of C1-inhibitor, C2, C3, C4 and factor B detected by ELISA and C2, C3 and factor B were shown to be functionally active. This study thus provides conclusive evidence that synthesis of complement components occurs locally within normal and inflamed synovial tissue. The local synthesis of complement within normal synovial joints may be of importance in their defence against infection, whereas in inflamed joints it may contribute to the inflammatory response.

Arthritis, Rheumatoid↗
PubMed1987

The role of ion channels and protein kinase C activation in the stimulation of complement protein synthesis.

Monocyte C2 synthesis is stimulated by antigen-antibody complexes (IC), carbamylcholine (C-Ch), phenylephrine (PE) and gamma-interferon. Tetrodotoxin or nifedipine abrogated the effects of IC, C-Ch and PE but did not influence the effect of gamma-interferon on C2 synthesis. Thus stimulation of C2 synthesis by IC, C-Ch and PE is dependent upon activation of Na+/K+ and Ca2+ channels, whereas gamma-interferon operates independently of these ion channels. Calcium channel agonists (CG28392 and BK8644) stimulated C2 synthesis, and this effect was prevented by nifedipine but not by tetrodotoxin. Thus Na+/K+ channels are activated prior to Ca2+ channels. Stimulation of C2 synthesis occurred when phospholipase C or phorbol myristate acetate (PMA) were added to the monocyte cultures, suggesting that PI cycle turnover and protein kinase-C (PK-C) activation are involved in the stimulation of C2 synthesis in monocytes. PMA, an activator of PK-C, stimulated the synthesis of C2, C3, B, P and C1-inhibitor approximately two-fold. In contrast gamma-interferon reduced synthesis of C3 and P by 44% and 22% respectively, and stimulated C1-inhibitor synthesis twelve-fold. These data suggest that the action of gamma-interferon complement synthesis is, at least partially, independent of PK-C activation. The effects of IC, C-Ch, PE, PI, CG28392, BK8644 and gamma-interferon were inhibited by trifluoperazine implying that calmodulin and/or other calcium binding proteins play a role in the modulation of complement protein production.

Calcium Channel Agonists↗
PubMed1986

Synthesis of complement components (C3, C2, B and C1-inhibitor) and lysozyme by human monocytes and macrophages.

The synthesis of C3, C2, factor B (B) C1-inhibitor and lysozyme has been studied in monocytes and macrophages isolated from the synovial fluids of patients with rheumatoid arthritis. Concentrations of all 5 proteins in culture supernatants were measured by the sandwich ELISA technique. Kinetic studies showed that only lysozyme and C3 could be detected in monocyte culture supernatants on the first day of culture, whereas C2, B and C1-inhibitor were not present until the third day. In contrast all 5 proteins could be detected in the supernatants of macrophage cultures on day 1. In both monocyte and macrophage cultures synthesis of lysozyme and C1-inhibitor continued throughout the culture period, whereas synthesis of C2, B and C3 appeared to be reduced after the fifth day in culture. Quantitative studies showed that the secretion rates of lysozyme (4,700 X 10(3) molecules/cell/hr) was similar in monocytes and macrophages. Synthesis rates for all 4 complement components in monocyte cultures were less than 0.2% of that for lysozyme. Although the synthetic rates were higher in macrophages, even then they constituted less than 2% of the rate for lysozyme. Synthetic rates for complement components, but not lysozyme, were increased by BSA-anti-BSA antigen-antibody complexes and reduced by serum-treated complexes. Although the functional activity of monocyte B was similar to that for serum, the activity of monocyte C2 was 5 times that of serum C2. As C42 formed with monocyte C2 had a half-life of 13.5 min at 30 degrees C, compared with 4.5 min for the enzyme formed with serum C2, it is probable that monocyte C2 is oxidized by the oxygen products of these cells.

Antigen-Antibody Complex↗
PubMed1985

Requirement for cations in the stimulation of C2 synthesis by human monocytes.

C2 synthesis by monocytes is stimulated by carbamylcholine acting on nicotinic receptors, phenylephrine acting on alpha 1 adrenergic receptors, and antigen-antibody complexes (IC) acting on Fc receptors. Stimulation of C2 synthesis is reversed by agents which block calcium (LaCl2, CoCl2, verapamil, nifedipidine, diltiazem) and sodium channels (tetrodotoxin) and calmodulin antagonists (trifluoperazine and W7). The changes in intracellular cyclic nucleotide levels that follow these receptor-ligand interactions (decreased cAMP, increased cGMP) do not occur in the presence of calcium and sodium channel blockers. These results suggest that the transmembrane signal which is involved in the stimulation of C2 synthesis is the entry of sodium and calcium ions. Whether this influx occurs by separate channels or a common channel has not been determined. The intracellular events involved in the stimulation of C2 synthesis appear to be calmodulin-dependent.

Antigen-Antibody Complex↗
PubMed1984

Cyclic nucleotides and their relationship to complement-component-C2 synthesis by human monocytes.

The time courses of changes in cyclic nucleotide levels in monocytes have been studied. Histamine and prostaglandin E2 (PGE2) produced a rapid rise in cyclic AMP (peak 15 min) levels, which returned to normal within 4h, whereas cholera toxin, NaF and phosphodiesterase inhibitors produced slow sustained rises lasting over 24h. With the exception of isobutylmethylxanthine (10 mumol X 1(-1), none of these reagents altered cyclic GMP levels. alpha 1-Adrenergic and nicotinic cholinergic receptor-ligand interactions and imidazole produced rapid and relatively short-lived falls in cyclic AMP, and rises in cyclic GMP. In contrast, prostaglandin synthetase inhibitors produced delayed but more sustained falls in cyclic AMP but no rises in cyclic GMP. Agents that increased cyclic AMP decreased complement-component-C2 production, and those that decreased cyclic AMP increased C2 production. Agents that increased cyclic GMP alone (ascorbate, nitroprusside and prostaglandin F2 alpha) did not affect C2 production. Antigen-antibody complexes that stimulate C2 synthesis produced falls in cyclic AMP and rises in cyclic GMP similar to those produced by adrenergic and cholinergic ligands. Serum-treated complexes and anaphylatoxins, which inhibited C2 production, were associated with changes in cyclic AMP similar to those produced by histamine and PGE2. These data suggest that there are two transmembrane signals involved in the regulation of C2 production by monocytes. The inhibitory signal is adenylyl cyclase activation. The stimulatory signal is not so obvious, but may be Ca2+ influx, since the time courses of changes in cyclic nucleotides produced by agents that stimulate C2 synthesis are identical, and alpha 1-adrenergic agonists cause the formation of Ca2+ channels.

Adenylyl Cyclases↗
PubMed1984

Adenosine A2 receptors on human monocytes modulate C2 production.

Adenosine inhibits C2 production by human monocytes. The use of synthetic analogues of adenosine suggested that the action was mediated by receptors of A2 type. EHNA which inhibits the enzyme adenosine deaminase and increases the concentration of intracellular adenosine also reduces C2 production. It is therefore possible that endogenous adenosine regulates C2 synthesis.

Adenine↗
PubMed1984

Role of C3 in the control of monocyte C2 production.

Serum-treated antigen-antibody complexes (IC) and DTSP-polymerized C3b and C3c inhibited the production of the second complement component (C2) by monocytes in culture, whereas monomeric C3, C3b, C3bi, C3c or C3d had no effect. The degree of inhibition of dithiobissuccinimidyl propionate (DTSP)-polymerized C3 was proportional to the degree of polymerization, dimer exhibiting less inhibitory activity than larger molecular weight polymers. The inhibitory effect of serum-treated IC and DTSP-polymerized C3b was abrogated by their pretreatment with Fab fragments of anti-C3, or pretreatment of monocytes with Fab fragments of antiserum to the C3b receptor (CR1). We have concluded that the inhibition of C2 production produced by serum-treated IC is due to bound C3b or C3bi, and is mediated by CR1. Furthermore cross linking of receptors is required for this effect; two or more than two receptors must be cross-linked for a significant effect to be observed.

Antigen-Antibody Complex↗
PubMed1983

Control of monocyte C2 production by cyclic AMP.

We have measured the changes in C2 production and in cyclic nucleotide levels in mononuclear leucocytes cultured in the presence of agents which change intracellular cAMP. Adenylcyclase activators, cholera toxin and phosphodiesterase inhibitors increased cAMP levels and reduced C2 production. Imidazole, hydrocortisone and prostaglandin synthetase inhibitors reduced cAMP and increased C2 synthesis. The level of cAMP after 2 hr incubation showed an inverse correlation with C2 levels in the culture supernatants. When histamine, which increases cAMP and reduces C2 production, and imidazole, which reduces cAMP and increases C2 synthesis, were added together, levels of cAMP and C2 did not differ significantly from those found in control cultures. On the basis of these observations we have concluded that changes in monocyte cAMP alter the production of C2.

Adenylyl Cyclases↗
PubMed1983

Anaphylatoxins inhibit C2 production.

Anaphylatoxins C5a and C3a and their des Arg derivatives inhibited C2 production by mononuclear phagocytes. C5a and C5adesArg which were approximately equipotent (IC50 = 10(-10) mol/l) were more effective than C3a (IC50 = 5 X 10(-8) mol/l) which was approximately 10-20-fold more potent than C3adesArg IC50 = 5 X 10(-6) mol/l). Inhibition of C2 production was only reversed slightly by the addition of either indomethacin or ETYA to the cultures. Intracellular levels of cAMP, were increased by anaphylatoxins. The level of cAMP showed a good inverse correlation with C2 levels in the culture supernatants. The data suggest that the reduction in C2 production produced by anaphylatoxins may be mediated by an increase in intracellular cAMP.

Anaphylatoxins↗
PubMed1983

Serum-treated antigen-antibody complexes inhibit the production of C2 and factor B by mononuclear phagocytes.

Antigen-antibody complexes enhanced the synthesis of C2 and factor B by human monocytes and macrophages, and C2 by guinea-pig macrophages. In contrast complexes that had been treated with serum inhibited the production of these components. The inhibitory effect of serum-treated complexes was abrogated by Fab fragments of anti-C3, anti-C3c and anti-C3d. It is therefore probable that inhibition was mediated by a C3 fragment bound to the complex. The enhancing effect of untreated complexes was reversible by serum-treated complexes, and the inhibitory action of serum-treated complexes was counteracted by untreated complexes. Such a system may be important in the regulation of the synthesis of complement components in response to local requirements.

Animals↗
PubMed1983

A comparison of the effects of heat-aggregated and chemically cross-linked IgG on monocyte C2 production.

Heat or alkali-aggregated IgG was found to inhibit C2 production by monocytes, whereas chemically cross-linked IgG and antigen-antibody complexes stimulated C2 synthesis. Chemically cross-linked IgG was shown to inhibit monocyte EA-rosette formation presumably because it blocked monocyte Fc receptors. Furthermore stimulation of C2 synthesis was limited to polymers of the IgG1 and IgG3 subclasses. In contrast, heat-aggregated IgG failed to inhibit monocyte EA-rosette formation significantly, and all the heat-aggregated IgG subclasses inhibited C2 production. It therefore appears that physically aggregated IgG does not bind effectively to Fc receptors. As the effects of physically aggregated IgG C2 production are similar to those of the hydrophobic proteins casein and alkali-denatured human serum albumin (HSA), it is suggested that hydrophobic residues in the aggregates bind preferentially to the lipid component of the cell membrane.

Animals↗
PubMed1982

Cyclic AMP mediated modulation of complement protein production.

We have investigated the mechanisms by which cAMP analogues and phosphodiesterase inhibitors, reduced the production of C2 by monocytes in culture. Pulse label studies with 3H-labelled aminoacids showed that dibutyryl cAMP (dbcAMP) impaired the secretion of newly synthesised protein, both total (acid-precipitable) and individual complement proteins (precipitated antibody by antisera to C4, C2, C3, C5, B, P, C3b inactivator and beta 1H). The intracellular degradation of newly synthesised protein was increased in dbcAMP-treated cultures and protein synthesis was reduced. Studies aimed at defining the temporal relationships between these changes showed that protein secretion was impaired on the first day of culture, and increased degradation of newly synthesised protein was obvious by day 2. Protein synthesis was not significantly reduced until day 3 of culture. It is proposed that changes in intracellular cAMP levels may act as a second signal in the control of protein production by monocytes.

Bucladesine↗
PubMed1982

Adrenergic receptors on monocytes modulate complement component synthesis.

The addition of adrenaline, noradrenaline or phenylephrine, but not isoprenaline to monocyte cultures enhanced synthesis of the second complement component (C2). This effect was abrogated by the concomitant addition of the receptor antagonist, phentolamine, but not the beta receptor antagonist propranolol. Thus the receptor involved is an alpha adrenergic receptor. Further studies showed that the receptor was of the alpha 1 subclass as prazosin inhibited the action of adrenergic agonists. Pulse label studies using 3H-amino acids showed that the enhancement of synthesis of eight complement components (C2, C3, C4, C5, factor B, properdin, beta 1H and C3b inactivator) and total protein synthesis were also increased. The possible mechanisms underlying these changes are discussed.

Adrenergic alpha-Agonists↗