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D A Lawrence

Publications and source records attributed to D A Lawrence.

At least 145 records · Page 8Linked to original sources

Lead-induced alterations of in vitro bone marrow cell responses to colony stimulating factor-1.

Bone marrow cell responsiveness to hematopoietic growth factors is an integral part of immune responsiveness. Since host resistance is often dependent on bone marrow cell responsiveness and Pb alters host resistance, the influence of Pb on bone marrow cell responsiveness to the hematopoietic growth factor CSF-1 was evaluated. Cell number, soft agar colony formation, cell cycle analysis, as well as 3H-thymidine incorporation were utilized to determine if CSF-1 driven bone marrow-derived macrophage (BMDM) proliferation in vitro is altered in the presence of PbCl2. Data obtained indicate that Pb potentiates the ability of CSF-1 to stimulate thymidine incorporation by BMDM; however, colony formation is inhibited reversibly, and the absolute number of cells in culture is adversely affected by Pb. Propagation of BMDM appears to be more sensitive to Pb (100 nM) than other immune parameters. The decrease in bone marrow cell responsiveness to CSF-1 in the presence of Pb observed in this system may contribute to the decrease in host resistance observed in Pb-exposed animals.

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Modification of lipoprotein patterns and retardation of atherogenesis by a fish oil supplement to a hyperlipidemic diet for swine.

We have studied the effect of addition of 30 ml cod liver oil (FO) daily to a highly atherogenic butter (BT) diet for swine on lesion development in the coronary arteries and aorta, plasma lipoprotein (LP) patterns, plasma levels of thiobarbituric acid-reactive substances (TBARS) and on tritiated thymidine-labeling indices ([3H]TdR LI) of smooth muscle cells (SMC) and monocyte/macrophages (M/M phi) in the atherosclerotic lesions. Seventeen male Yorkshire swine (11.1 +/- 0.4 kg) were divided into 3 groups: BT (n = 6), BT + FO (n = 6) and mash (n = 5). They were fed the respective diets for 4 months. Terminally, fasting plasma was obtained and cholesterol contents were determined in various fractions of lipoproteins separated by density gradient ultracentrifugation, Pevikon block electrophoresis and immunoelectrophoresis. Apoprotein (B, A-I, E and C) contents of the plasma and lipoprotein fractions were determined by polyacrylamide gel electrophoresis and densitometry of gels stained with Coomassie blue. Swine were injected intramuscularly with 0.5 mCi/kg of [3H]TdR 2 h before death. The aorta and coronary arteries were perfusion fixed in situ under anesthesia. Samples were obtained for microscopic morphometry, autoradiography and immunohistochemistry from distal abdominal aorta, thoracic aorta, and proximal coronary arteries; left main (LM), left anterior descending (LAD), left circumflex (LCX), right main (RM), and right coronary artery (RCA). On the BT diet without FO there was extensive atherosclerotic (AS) lesion development, which was drastically reduced by the addition of FO to the BT diet in all sites by from 71 to 94%. The overall plasma cholesterol (CH) levels were reduced only modestly by the FO (816 +/- 64 to 629 +/- 14 mg/dl) but the distribution of CH in the various lipoprotein classes was remarkably altered. The CH in the large lipoprotein molecules containing both B and E apoproteins was reduced from 488 +/- 84 to 204 +/- 17 mg/dl by the FO with an almost corresponding increase in the conventional LDL molecules containing apo B only (158 +/- 29 to 344 +/- 15 mg/dl). We offer the hypothesis that the large apo B,E containing molecules are much more atherogenic than the smaller apo B containing molecules. This hypothesis is supported by a highly significant correlation between extent of lesion development in all arterial sites and plasma levels of CH in apo B,E containing lipoproteins. Plasma TBARS were elevated by the BT + FO diet but seemed to have no significant effect on the lesions.(ABSTRACT TRUNCATED AT 400 WORDS)

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The inhibitory effects of N-ethylmaleimide, colchicine and cytochalasins on human T-cell functions.

The thiol-alkylating agent, N-ethylmaleimide (NEM), was found to inhibit the response of human peripheral blood mononuclear cells to the T-cell mitogen, concanavalin A (Con A). NEM (10 microM) blocked Con A-induced agglutination, production of interleukin-2 (IL-2) and expression of the IL-2 receptors (Tac) without toxicity. In order to determine whether the effects of NEM on lymphokine production were related to inhibition of agglutination, we compared the immunosuppressive effects of NEM with those of cytochalasin A, cytochalasin B and colchicine. NEM did not inhibit E-rosette (ER) formation, suggesting that it does not interfere with actin filaments. Low concentrations of NEM (4 microM) inhibited IL-2 production and Tac expression without inhibiting agglutination, while 6-10 microM NEM blocked agglutination and DNA synthesis as well. In contrast, 5-10 microM cytochalasin B (CB) inhibited ER formation, agglutination, Tac expression and DNA synthesis, but augmented IL-2 production by three- to ten-fold. Colchicine (0.1-10 microM) had no effect on ER formation or agglutination and augmented IL-2 production by as much as 18-fold. However, colchicine blocked Tac expression by greater than 40% and DNA synthesis by greater than 80%. Cytochalasin A (CA), which has the thiol-reactive properties of NEM, the actin filament-disrupting properties of CB, and the microtubule-disrupting properties of colchicine, exhibited the immunosuppressive effects of all three compounds. These studies suggest that the inhibitory effects of NEM on IL-2 production do not appear to be due to reactivity with the cytoskeleton, but are probably due to effects on signal transduction pathways leading to IL-2 production and expression of IL-2 receptors.

Agglutination↗

Bifunctional activity of transforming growth factor type beta on the growth of NRK-49F cells, normal and transformed by Kirsten murine sarcoma virus.

Transformation of rat NRK-49F cells (49F) by Kirsten murine sarcoma virus (Ki-MSV) renders these cells (Ki-49F cells) capable of autonomous anchorage independent (AI) growth. As compared to nontransformed 49F cells, the transformation by Ki-MSV does not modify the cell response to transforming growth factor-beta (TGF-beta) in monolayer conditions, but alters it in A I growth conditions. The growth of nontransformed or Ki-MSV-transformed adherent 49F cells is slowed down by porcine TGF-beta, and this effect is reversed by epidermal growth factor (EGF). This decrease in the cell growth rate, induced by TGF-beta, does not affect the cloning efficiency of untransformed and transformed adherent 49F cells. Contrarily, porcine TGF-beta decreases the A I cloning efficiency of Ki-49F cells in agar-gelled medium; this effect is only partly reversed by EGF, which does not synergise with TGF-beta to enhance the A I growth as in the case of untransformed 49F cells. Media conditioned by 49F cells, Ki-49F cells, and chicken embryo fibroblasts contain a latent TGF-beta whose capacity to promote the A I growth of 49F cells and to inhibit that of Ki-49F cells is unmasked by acidification. The same situation exists concerning TGF-beta from human platelets. Neutral extracts are inefficient in both tests of promotion and inhibition of A I growth and contain an acid-activable component with an apparent molecular weight of 600 kd. In acid extracts, a 5-9 kd apparent molecular weight component is responsible for the A I growth enhancement of 49F cells and the A I growth inhibition of Ki-49F cells. Further purification by reverse phase chromatography shows that both activities strictly coelute at the same point (32%) of an acetonitrile gradient. These results indicate that TGF-beta is present in physiological conditions as a latent form which requires activation for inhibiting the A I growth of transformed cells as well as for enhancing that of 49F cells.

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Effect of lead on macrophage function.

Lead (Pb) has been shown to alter various parameters of immune function such as host resistance and antibody formation. In addition, various heavy metals have been implicated as inducers of autoimmunity. In these experiments, macrophages, isolated from the peritoneal cavity of mice exposed to various doses of lead in vivo as well as cells exposed in vitro were tested for the following immunologic parameters: phagocytosis, antigen presentation, interleukin 1 production, and their ability to stimulate the autologous mixed lymphocyte reaction (AMLR). The results obtained indicate that Pb appears to alter the ability of macrophages to present antigen by enhancing the AMLR while having no effect on phagocytosis or IL-1 production. These data suggest that Pb may interfere with antigen-specific interactions between macrophages and T cells.

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Four sulfhydryl-modifying compounds cause different structural damage but similar functional damage in murine lymphocytes.

Four thiol-modifying compounds were used to inhibit murine lymphocyte mitogenesis. The compounds were a copper sulfate/O-phenanthroline complex (CuP) to oxidize surface thiols, N-ethyl maleimide (NEM) to alkylate surface and intracellular thiols, D,L-buthionine-S,R-sulfoximine (BSO) to prevent synthesis of glutathione, and hydrogen peroxide, which reacts with various cellular constituents, including sulfhydryls. Splenic lymphocytes were incubated with one of the four compounds, washed, and then stimulated with the B cell mitogen, LPS, or the T cell mitogen, Con A. In spite of their differing chemical reactivities and differing effects on cell viability, lipids, and total, protein, and non-protein thiols, the four sulfhydryl-modifying compounds had very similar effects on the kinetics and inhibition of lymphocyte growth. All compounds had complex effects on mitogenesis, causing enhanced, delayed, or inhibited tritiated thymidine incorporation. Although the total thiol contents of untreated T cells and B cells were found to be equivalent, the LPS response consistently was inhibited by lower concentrations than the Con A response, suggesting that B cells were more sensitive than T cells to thiol modification. To compare compounds the efficiency of inhibition was determined by functionally relating reductions in mitogenesis with reductions in thiol content of the cells. The compounds differed in inhibitory efficiency; thus, damage to some thiols must be more important than damage to others. CuP ablated mitogenesis with the least change in thiol content. Therefore, surface sulfhydryls appear critical in lymphocyte mitogenesis. With all compounds inhibition of mitogenesis occurred over a very narrow range of thiol content, suggesting that the thiols important in inhibition were few in number relative to the total thiol content of the cell.

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Antagonistic action of RU38486 on the activity of transforming growth factor-beta in fibroblasts and lymphoma cells.

Transforming growth factor-beta (TGF-beta) is a multifunctional protein involved in the control of proliferation, differentiation and other functions in many cell types. The anchorage-independent growth of some established lines of untransformed fibroblasts in soft agar is induced by TGF-beta and requires in addition exogenous EGF for certain target cells, notably rat NRK-49 cells. The formation of colonies of NRK-49F cells is completely inhibited by the synthetic 11-beta substituted nor-steroid RU38486 added at a final concentration of 1.3 X 10(-5) M. We also explored the effect of TGF-beta on Daudi and Raji lymphoma cells by measuring the production of Epstein-Barr Virus (EBV) early antigens (EA). In Daudi cells an induction capacity giving rise to 10-16% positive EA-cells was observed; in Raji cells the induction only reached between 6 and 8%. The induction was partially inhibited by the anti-steroid RU38486 in both systems. Thus, RU38486 not only antagonizes the glucocorticoid hormone action but also interferes with the effects of TGF-beta in fibroblasts and in lymphoma cells. The molecular basis of the interactions observed was investigated by considering (1) the binding to specific receptors, (2) transfection experiments, in order to examine if the interference of the anti-steroid with TGF-beta activities occurs at the transcriptional level as in the case of glucocorticoid induction. The results suggest that the blocking by antiglucocorticoids of the effects of TGF-beta and glucocorticoids, in fibroblasts and lymphoma cells, occurs by different mechanisms.

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Immunomodulation of in vitro antigen presentation by cations.

The ability of various cations to alter an antigen-specific (ovalbumin) T cell activation system in vitro has been assessed. This in vitro system includes analysis of antigen capture, processing, and presentation by antigen presenting cells (APC) (splenocytes, B cells or macrophages) in a major histocompatibility complex (MHC) restricted fashion and the evaluation of the activation of a T-cell hybridoma, DO-11.10, which is specific for "processed" ovalbumin in association with I-Ad. Activation is determined by production of IL2 which is quantitated in a bioassay with HT-2 cells. Numerous metals (10 and 100 microM) were screened in the coculture assay (APC and DO-11.10 plus ovalbumin). Metals with inhibitory effects were Cd greater than Cu greater than Pb greater than Zn. Co and Cr had no modulatory effect and Ni had an enhancing effect (increased IL2 production). The effects of the modulatory metals were further assessed for influences on the individual cellular components of this system. Cd was toxic to all cell types whereas Cu was toxic only to irradiated splenocytes and Do-11.10. Pb was the only metal which was not toxic to any cell type but still inhibited antigen presentation. To prevent influences of metals on DO-11.10 or carryover into the bioassay, APC were preincubated with metals and then assessed for presentation capability. After preincubation, only Cd, Pb, and Cu were inhibitory. The inhibition by Cd was due to toxicity. Cu inhibited only irradiated splenocyte presentation and this effect was due to toxicity. Pb inhibition was due to a mechanism other than toxicity and its biochemical influences on APC are discussed.

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The effect of metals on IL-2-related lymphocyte proliferation.

The heavy metals Pb, Ni and Zn have previously been demonstrated to stimulate the proliferation of an Lyt1+2-, L3T4+T-cell. This proliferation required the presence of Ia+ cells and was blocked by monoclonal antibodies directed against self I-A, self I-E and L3T4a. The work reported here examined the role of T-cell factors (IL-2 and gamma-IFN) and their receptors in the metal-induced lymphoproliferation. The metals Pb, Ni and Zn, at concentrations (100 microM) which stimulate T-cell proliferation, had little effect on the ability of the IL-2-dependent cell line HT-2 to respond to exogenous IL-2. This suggests that Pb, Ni and Zn do not modulate the ability of IL-2 to interact with the IL-2 receptor. Ni and Zn significantly enhanced the synthesis/secretion of IL-2 by cultured splenocytes and the expression of the receptor for IL-2; however, Pb produced only slight enhancement. Likewise, anti-IL-2 receptor (7D4) antibodies were able to inhibit a significant portion of the Ni- and Zn-, but not Pb-, induced lymphoproliferation. The residual 3H-thymidine incorporation observed in the presence of anti-IL-2 and anti-IL-2R may represent cycling B-cells induced to proliferate by activated, but non-cyclin, T-cells. Monoclonal anti-gamma-IFN (R4/6A2) equally inhibited all metal-induced lymphoproliferation, suggesting that metal-induced lymphoproliferation is dependent on the induction of gamma-IFN as well as IL-2 synthesis. The Pb-induced response being the least dependent on IL-2 lends support to the hypothesis that Pb, Ni and Zn may activate T-cells and/or B-cells by different mechanisms.

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Chemically and virally transformed cells able to grow without anchorage in serum-free medium: evidence for an autocrine growth factor.

BA10-IR transformed cells, obtained by treating Syrian hamster embryo fibroblasts (HEF) with 7-methylbenz(a)anthracene and cultivated for a long period, are highly tumorigenic and grow in suspension as aggregates (spheroids) (Levy et al., 1976). They also grow in attached form or as spheroids in serum-free (S-) synthetic medium, without insulin and transferrin, and form anchorage-independent (AI) colonies in this same, but semi-solid, medium. This exceptional phenotype was acquired stepwise, after other transformation parameters, and appears to be related to the capacity of the transformed cells to respond to a mitogenic growth factor which they secrete. The response to this autocrine factor is amplified by insulin and transferrin. Untransformed HEF, at late and early passages, and also mouse and rat embryo fibroblasts, secrete factors equally active on BA10-IR cells; but HEF do not respond, in S- medium, to their factor, or that of BA10-IR cells. Rat FR3T3 fibroblasts transformed by Kirsten murine sarcoma virus (FR3T3-Ki cells) also form AI colonies in semi-solid S- medium, secrete an autocrine factor potentiated by insulin and transferrin, and respond to the factors active on BA10-IR cells. However, they form far fewer colonies without additives, and respond as well to the mitogenic factors only in the presence of insulin and transferrin. BA10-IR cells and FR3T3-Ki cells also release beta-TGF, or a related factor, in an active and a latent form, activable by acidification, and HEF latent, activable beta-TGF. However, the factors shed by BA10-IR cells or HEF which stimulate AI growth of BA10-IR and FR3T3-Ki cells are proteins which seem unrelated to known transforming growth factors. Two major cellular alterations characteristic of the transformed phenotype in vitro are the ability to grow in the absence of anchorage, in semi-solid medium, and reduced dependence on serum growth factors (Hanafusa, 1977; Tooze, 1980). These alterations are often expressed together, and anchorage independence also appears to be the in vitro transformation parameter which correlates best with the tumorigenicity of the transformed cells (Pollack et al., 1975; Shin et al., 1975; Cifone and Fidler, 1980). However, this correlation is not constant (cf., Tooze, 1980). The cellular changes which confer anchorage independence remain unknown, but the culture conditions which allow anchorage-independent (AI) growth are better known. This growth occurs in the same media which permit the growth of attached cells, but generally requires serum.(ABSTRACT TRUNCATED AT 400 WORDS)

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Transdifferentiated embryonic neuroretina cells: an in vitro system to study crystallin aggregation process.

Transdifferentiated embryonic quail neuroretina cells synthesize in vitro crystallins (the lens-specific proteins) and form lentoid bodies (structures that mimic lens fiber cells) which also contain crystallins. A comparative study on the size of crystallins is reported in 7-day-old embryonic quail lenses, in 7-day-old embryonic quail transdifferentiated neuroretina cells (normal and MH2 transformed), and in isolated lentoid bodies. Analyses are performed using Superose FPLC in combination with SDS-PAGE and Western blot procedures. In quail lenses, an apparent 560-580-kDa alpha crystallin homopolymer is found and delta crystallin, the major avian lens protein, is detected as a 180-kDa tetramer. beta Crystallins, present in low amount within the 180-kDa peak, are a heterogeneous population composed of subunits of molecular weight identical to those found in chick lenses. In addition, an apparent 46-kDa monomeric delta crystallin is found. Normal and MH2-transformed neuroretina cultures produce an alpha crystallin polymer of lower molecular weight (450 kDa) and delta crystallin in a monomeric or dimeric form. The Western blot pattern of beta crystallins from MH2-transformed neuroretina cultures is strictly identical to that of quail lens beta crystallins. In particular, the beta B1 crystallin, which is specific to lens fiber cell differentiation, and the major beta 25-kDa crystallin are present. However, analysis of isolated lentoid bodies from normal transdifferentiated quail neuroretina cultures showed alpha and delta crystallins of comparable size to those found in lens extract, in particular the delta crystallin in tetrameric form. The lentoid body lens-like structure could favour the crystallin aggregation process.

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Inactivation of plasminogen activator inhibitor by oxidants.

The rapidly acting plasminogen activator inhibitor (PAI) purified from cultured bovine aortic endothelial cells (BAEs) was inactivated during iodination with chloramine T and other oxidizing iodination systems. Inactivation was observed in the absence of iodine, suggesting that the loss of activity resulted from the oxidizing conditions employed. In an attempt to further study the nature of this inactivation, the PAI was treated with chloramine T under conditions that specifically oxidize methionine and cysteine residues. Both PAI inhibitory activity and the ability of the PAI to form complexes with tissue-type PA were decreased in a dose-dependent manner by such treatment. The PAI was more sensitive to oxidative inactivation than urokinase, elastase, and alpha 1-protease inhibitor. Incubation of the chloramine T inactivated PAI with methionine sulfoxide peptide reductase in the presence of dithiothreitol (DTT) restored more than 90% of the PAI activity. The reductase is a DTT-dependent enzyme that specifically converts methionine sulfoxide to methionine. Little activity was restored by either the reductase or DTT alone. These results indicate that the oxidation of at least one critical methionine residue is responsible for the loss of PAI activity upon iodination. In this respect, the BAE PAI resembles alpha 1-protease inhibitor, a well-characterized elastase inhibitor that also is inactivated by oxidants. Both inhibitors are members of the serine protease inhibitor superfamily (Serpins), and both have a methionine residue in their reactive center.

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Beta-transforming growth factor is stored in human blood platelets as a latent high molecular weight complex.

Human blood platelets, the richest known source of beta-transforming Growth Factor extractable under acid conditions, release in neutral extracts (pH 7.2) a latent form of this growth factor with an apparent molecular weight of 400 Kd. This latent form, poorly active on rat NRK-49F indicator cells in soft agar assays can be activated by exposure to acid pH or 8 molar urea. The acid activated beta-Transforming Growth Factor from neutral extracts elutes on Biogel P60, in 1 molar acetic acid, as a broad peak of apparent molecular weight 15-30 Kd, like when this factor is extracted from platelets by the usual acid-ethanol procedure. Moreover, beta-Transforming Growth Factor from both acid activated neutral extracts and from acid-ethanol extracts elutes on reverse phase at 30% acetonitrile. We suggest that beta-Transforming Growth Factor is stored in human blood platelets as a poorly active high molecular weight complex which may be dissociated and activated in appropriate in vivo microenvironments.

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Cell surface and cell cycle analysis of metal-induced murine T cell proliferation.

The heavy metal cations Pb2+, Ni2+ and Zn2+ have previously been shown to induce T cell proliferation which required the presence of both T cells and Ia+ cells at the initiation of culture. This work has examined the ability of these metals to induce cell cycle entry as determined by acridine orange cell cycle analysis. Cell surface phenotype analysis, performed on splenocytes stimulated with optimum metal concentrations (100 microM), indicated that in vitro T cell recovery (growth and/or longevity) was enhanced by Pb2+, Ni2+, and Zn2+. Furthermore, simultaneous examination of cell surface phenotype and cell cycle progression (propidium iodide) indicated that the predominant cell type proliferating in response to these metals was Thy-1.2+. The metals differentially induced L3T4+ and Lyt-2+ cells to enter the cell cycle. The ability of various monoclonal antibodies to modulate metal-induced proliferation was examined. Anti-L3T4a, anti-I-A and anti-I-E blocked metal-induced proliferation. Anti-Lyt-2 only partially inhibited whereas anti-Lyt-1 was stimulatory. These results suggest that recognition of major histocompatibility complex-encoded class II molecules is required for the induction of proliferation by these metals (similar to the autologous mixed lymphocyte response).

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Fluorometric quantitation of cellular and nonprotein thiols.

A microfluorometric assay for thiols has been developed using the thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM). The technique may be used to quantitate either cellular or plasma thiols over a range of 0.01 to 3.0 nmol and may be used with as few as 1-3 X 10(5) cells giving highly proportional and reproducible results. Values for nonprotein thiols obtained with this assay agree well with previous reports on glutathione (GSH) levels for both lymphocytes and plasma. Readings are determined with the aid of an automated fluorescence microplate reader which allows up to 96 samples, including standards, to be read at the same time. Cellular thiols accessible after lysis were also quantitated before and after treatment of intact cells with various thiol-reactive chemicals. Interestingly, HgCl2, bromoethanesulfonic acid, and N-ethylmaleimide differentially modified protein and nonprotein thiol levels.

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Stimulation of murine lymphocyte responses by cations.

The capacity of the heavy cations Pb, Ni, and Zn to modulate murine in vitro lymphocyte responses was examined. Pb and Ni (100 microM) were shown to enhance the in vitro plaque-forming cell (PFC) response to sheep red blood cells while 100 microM Zn had inhibitory effects. Each metal was able to stimulate the proliferation of murine splenocytes as determined by [3H]thymidine incorporation and autoradiography. The enhancing effect of the metals on the PFC response was observed whether the results were expressed on a per culture or a per cell basis, indicating an actual increase in B-cell differentiation. Both the PFC response and the proliferative response were shown to be sensitive to the type of medium employed (M-199 gave optimum results) and to the presence or absence of 2-mercaptoethanol. As in autologous mixed-lymphocyte responses peak proliferation occurred after Day 5 in culture, was cell density dependent, and required the presence of both T cells and Ia+ cells. Treatment of splenocytes with anti-Thy-1.2, anti-Lyt-1, or anti-L3T4 plus complement completely abrogated the proliferative response, indicating that a Lyt-1+, Lyt-2-, L3T4+ T-cell was required for the induction of proliferation. The data are consistent with the hypothesis that the metals are capable of modifying the immune response directed at self either by directly altering self constituents (class II) or by modulating the autologous T-cell response.

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