John L. Gordon

TNFR-Associated Factor Family Protein Expression in Normal Tissues and Lymphoid Malignancies

TNFR-associated factors (TRAFs) constitute a family of adapter proteins that associate with particular TNF family receptors. Humans and mice contain six TRAF genes, but little is known about their in vivo expression at the single cell level. The in vivo locations of TRAF1, TRAF2, TRAF5, and TRAF6 were determined in human and mouse tissues by immunohistochemistry. Striking diversity was observed in the patterns of immunostaining obtained for each TRAF family protein, suggesting their expression is independently regulated in a cell type-specific manner. Dynamic regulation of TRAFs was observed in cultured PBLs, where anti-CD3 Abs, mitogenic lectins, and ILs induced marked increases in the steady-state levels of TRAF1, TRAF2, TRAF5, and TRAF6. TRAF1 was also highly inducible by CD40 ligand in cultured germinal center B cells, whereas TRAF2, TRAF3, TRAF5, and TRAF6 were relatively unchanged. Analysis of 83 established human tumor cell lines by semiquantitative immunoblotting methods revealed tendencies of certain cancer types to express particular TRAFs. For example, expression of TRAF1 was highly restricted, with B cell lymphomas consistently expressing this TRAF family member. Consistent with results from tumor cell lines, immunohistochemical analysis of 232 non-Hodgkin lymphomas revealed TRAF1 overexpression in 112 (48%) cases. TRAF1 protein levels were also elevated in circulating B cell chronic lymphocytic leukemia specimens (n = 49) compared with normal peripheral blood B cells (p = 0.01), as determined by immunoblotting. These findings contribute to an improved understanding of the cell-specific roles of TRAFs in normal tissues and provide evidence of altered TRAF1 expression in lymphoid malignancies.

Effects of neutrophil elastase and other proteases on porcine aortic endothelial prostaglandin I2 production, adenine nucleotide release, and responses to vasoactive agents.

The effects of neutrophil elastase on endothelial prostacyclin (PGI2) production, nucleotide release, and responsiveness to vasoactive agents were compared with the effects of cathepsin G (the other major neutral protease of neutrophils), pancreatic elastase, trypsin, chymotrypsin, and thrombin. PGI2 production by pig aortic endothelial cells cultured on microcarrier beads and perfused in columns was stimulated in a dose-dependent manner by trypsin, chymotrypsin, and cathepsin G (1-100 micrograms/ml for 3 min). Thrombin, while active at low concentrations (0.1-10 National Institutes of Health U/ml), induced smaller responses. Neutrophil and pancreatic elastase had little or no effect on PGI2 production. Dose-dependent, selective release of adenine nucleotides was induced by neutrophil elastase (3-30 micrograms/ml). The other proteases were much less active; for example, trypsin (100 micrograms/ml) induced a response only approximately 5% as great as did 30 micrograms/ml neutrophil elastase. After exposure to 30 micrograms/ml neutrophil elastase, cells did not exhibit the characteristic burst of PGI2 production in response to extracellular ATP; responsiveness gradually returned after 40-120 min. This effect was not seen with the other proteases. Elastase partly inhibited responses to bradykinin and had no effect on PGI2 production that was stimulated by ionophore A23187. There was no evidence of cytotoxicity, as measured by release of lactate dehydrogenase. Neutrophil degranulation can generate concentrations of elastase and cathepsin G comparable with those tested in the present study, and the effects of these enzymes on endothelial function lead us to suggest that they may play a role in vasoregulation and vascular pathology.

P2 purinoceptors in the blood vessel wall.

Commentaire sur les recents progres concernant la regulation de la reponse vasculaire par les nucleotides adenyles extracellulaires. Mise au point des connaissances sur les purinorecepteurs P2 de la paroi des vaisseaux sanguins

CD5-positive and CD5-negative human B cells converge to an indistinguishable population on signalling through B-cell receptors and CD40

Whether CD5 on B cells marks a subset functionally distinct from the conventional CD5 negative (CD5neg) adult population or is more an indicator of activation, remains contentious. Here we have investigated whether CD5 positive (CD5pos) and CD5neg B cells can be distinguished in terms of their response to surrogate signals aimed to model, in vitro, T‐cell dependent (TD) and T‐independent (TI) encounters with antigen in vivo: the predominantly CD5pos B‐cell population found in cord blood, CD5 B cells positively selected from tonsils and their CD5neg counterparts, were compared. Neonatal B cells displayed a near‐identical phenotype to that of adult CD5pos B cells, being characterized by uniform immunoglobulin M (IgM), immunoglobulin D (IgD), CD23 and CD44 coexpression. When cultured with anti‐IgM maintained at high density on CD32‐tranfected mouse L cells to model TI responses or on CD40 ligand (CD40L)‐bearing L cells (with or without captured anti‐IgM) to model TD encounters, DNA synthesis was stimulated to a similar extent in all three populations. Focusing on CD5 and CD23, we found that – although the signals delivered promoted distinct profiles of expression – under each condition of activation, the phenotypes that emerged for adult CD5pos and CD5neg B cells were remarkably similar. Neonatal B cells displayed a greater diminution in CD5 expression than adult CD5pos B cells following CD40 signals but otherwise the two populations again behaved similarly. The inclusion of interleukin‐4 (IL‐4) to cultures where cells were costimulated via surface (s)IgM and CD40 resulted in a complete loss of CD5 expression and a corresponding hyperexpression of CD23, irrespective of the population studied. The near‐identical response of CD5pos and CD5neg B cells to surrogate TD or TI signals in vitro and their convergence to indistinguishable phenotypes is wholly supportive of CD5 being a fluctuating marker of activation rather than it delineating functionally distinct subsets.

Desensitization of agonist-stimulated prostacyclin release in human umbilical vein endothelial cells

Prostacyclin (PGI2) release was studied in perfused columns of human umbilical vein endothelial cells cultured on microcarrier beads. Substantial homologous desensitization of PGI2 release occurred when cells were exposed to agonist for 2 min after a previous exposure; the extent depended on the concentration and duration of the first challenge. Recovery from exposure to ATP or bradykinin was complete in less than 80 min; recovery from thrombin was incomplete after greater than 80 min, and this was apparently related to its proteolytic activity. Experiments with ibuprofen, a reversible inhibitor of cyclo-oxygenase, demonstrated that homologous desensitization did not involve inactivation of cyclo-oxygenase. ATP and bradykinin did not induce heterologous desensitization. Thrombin and trypsin induced cross-desensitization, but neither agonist significantly reduced responses to ATP or bradykinin, suggesting that a common proteolytic mechanism is responsible for their ability to induce PGI2 synthesis. We conclude that desensitization of PGI2 release in response to physiological agonists is generally agonist-specific and involves modulation of molecular events at or close to the receptors involved, rather than inactivation of prostanoid biosynthesis.

Adenosine Uptake and Adenine Nucleotide Metabolism by Vascular Endothelium

Recent research has emphasized that extracellular purines (particularly adenosine and A TP) may be important regulators of vessel tone, commonly producing vasodilation, through their binding to receptors currently classified as P1 (adenosine) and P2 (ATP) [1]. The direct control of vascular tone has usually been thought of in terms of the action of mediators at receptors on vascular smooth muscle cells, causing contraction or relaxation. However, within the last two years it has been recognized that endothelial cells are necessary modulators of the vasodilator response to several agents, including ATP [2–4]; before the publication of these results, such a role for endothelium in the control of vascular tone had been largely unconsidered. Indeed, until fairly recently, the concept that endothelial cell functions could contribute in any way to the regulation of blood flow, for example, by the metabolism of circulating vasoactive compounds, was not widely recognized.

Monoamine oxidase activities of porcine vascular endothelial and smooth muscle cells

Abstract Amine uptake by cultured vascular cells was studied under conditions minimizing nonenzymic oxidation. 5-Hydroxytryptamine (5HT) was accumulated only very poorly; detailed kinetic analysis couid not be performed, but there was no evidence for a saturable high affinity process. Comparison of β-phenylethylamine (PEA) and 5HT metabolism in intact cells and lysed cells demonstrated that the rates of entry of the amines into cells usually limited their metabolism especially at low (μM) concentrations. Primary cultures of aortic endothelial cells metabolised 5HT and PEA substantially faster than did subcultured endothelium. Subcultured aortic vascular smooth muscle cells and endothelial cells metabolised PEA and 5HT with comparable specific enzyme activities to those found in aortic medial tissue. Inhibition by clorgyline of PEA, 5HT and benzylamine (BZA) metabolism reveaied, however, that while aortic tissue possessed monoamine oxidase (MAO) types A and B and a comparable amount of a clorgyline resistant amine oxidase(s) (CRAO), cultured vascular cells possessed MAO-A, but little or no CRAO or MAO-B. Cultured venous endothelium, and smooth muscle from several vascular sites, metabolised PEA and 5HT at similar rates to those found in aortic cells. the studies demonstrate that although cultured porcine endothelial and smooth muscle cells from large blood vessels contain MAO, they do not apparently possess the amine transport process present in the lung. Additionally, conditions of culture can affect both the extent of amine metabolism and the pattern of amine oxidase present.

Stimulation of endothelial cells by protease activity in commercial preparations of xanthine oxidase.

The oxygen radical generating system of xanthine oxidase plus xanthine, which has been used as a model for the oxidative burst of activated granulocytes, is known to damage endothelium in vivo and in vitro. We previously observed effects (inhibited by catalase, and thus associated with the formation of H2O2) on several parameters of endothelial function, using a non-commercial preparation of xanthine oxidase. Our present study demonstrates that xanthine oxidase from two different commercial sources has additional effects on endothelial morphology and ion flux that are substrate-independent (i.e. produced in the absence of added xanthine) and are attributable to the presence of pancreatin (a crude enzyme mixture used in the commercial preparation of xanthine oxidase from milk). These effects are related to the tryptic activity of pancreatin and extend previous observations on the effects of neutral proteases on endothelial cells. Our results emphasise the practical point that studies on the effects of commercial xanthine oxidase preparations on endothelial cells must take account of their trypsin-like activity as well as their capacity to generate oxygen products.

Metabolism of serotonin and adenosine

The major role played by endothelial cells in the inactivation of a wide variety of vasoactive and platelet-active compounds is becoming apparent as our knowledge of endothelial biology increases, mainly because of the ability to isolate and culture these cells. Endothelium is currently believed to be responsible for the inactivation of circulating thrombin, bradykinin, several prostaglandins and amines, adenosine and purine nucleotides. In this chapter we shall discuss the metabolism by endothelial cells of three of these molecules: serotonin (5-hydroxytryptamine, 5HT), adenosine and ATP. 5HT is released into the plasma when platelets are activated. It is a powerful vasoconstrictor in most vascular beds, although in some species it is a vasodilator in certain small vessels (1). Virtually all the 5HT in the blood is carried by the platelets in dense storage granules; it is released in common with other granule contents when platelets are stimulated by a variety of agents (e.g. ADP, thrombin, collagen) and is itself a platelet aggregating agent (2).

Influence of human β-thromboglobulin on prostaglandin production by pig aortic endothelial cells in culture

One batch of human β-thromboglobulin (βTG) was found to inhibit PGI2 production by pig aortic endothelial cells in culture. PGE2 production by these cells was also inhibited. βTG had a similar inhibitory effect on prostaglandin production by pig aortic smooth muscle and mouse 3T3 cells and this inhibitory activity was reduced to 50% of control value on Amicon filter dialysis. Three subsequent batches of βTG were found to be without effect on prostaglandin production by aortic endothelium and 3T3 cells. Ammonium acetate and sodium azide, which are used during the preparation of βTG, were tested for inhibitory activity and were active against endothelial cell prostaglandin production only at concentrations that were cytotoxic; these ions were therefore unlikely to account for the inhibitory activity of βTG initially seen. Freshly isolated endothelial cells and subcultured endothelial and smooth muscle cells at different stages of growth to confluence were also tested to see if the previously described βTG receptor had been lost during growth of cells in culture. The effect of γTG or low affinity platelet factor 4 (LA-PF4), the precursor of βTG, was compared with that of βTG. No inhibitory effects of βTG or γTG were found under these conditions. We therefore conclude firstly, that any effects of βTG or γTG on prostaglandin production by vascular cells in culture cannot be reproducibly demonstrated, and secondly, that any such effect is not specific for PGI2 or for endothelium.