Jean Favero

Ecto-5′nucleotidase and adenosine deaminase activities of lymphoid cells

Ecto-5′nucleotidase and adenosine deaminase activities were determined in mouse lymphoid populations and in 18 established human T, B, or null lymphoblastoid cell lines. We showed that correct determination of 5′nucleotidase in lymphoblasts necessitated the use of 5′[32P]AMP as substrate and that previously reported data, obtained with 5′AMP radiolabeled on the adenosine moiety, had to be reevaluated. We confirmed the hypothesis that 5′nucleotidase and adenosine deaminase levels are linked to the differentiation stage of mouse lymphocytes. An inverse relationship appeared between these two enzymatic activities. A correlation was found between the ratio of 5′nucleotidase versus adenosine deaminase activities and some characters currently used to define the stage of lymphoid cell maturation. A similar relationship was found between 5′nucleotidase and adenosine deaminase activities of human lymphoblastoid cell lines: their ratio displayed a good correlation with their terminal deoxynucleotidyl transferase activity. As a consequence we present the hypothesis that the ratio of 5′nucleotidase versus adenosine deaminase activities might be related to the maturation stage of lymphoid cells.

Involvement of CD4 in interleukin-6 secretion by U937 monocytic cells stimulated with the lectin jacalin

The lectin jacalin is mitogenic for CD4 expressing T lymphocytes, interacts with the CD4 molecule, and inhibits HIV infection of CD4+ cells. In the present study the effect of jacalin was tested on cells from the monocyte/macrophage lineage that also express the CD4 molecule. We used CD4+ promyelomonocytic U937 cells differentiated towards the monocytic/macrophage lineage with either a mixture of two physiological agents, retinoic acid (RA) and 1α,25‐dihydroxyvitamin D3 (VD), or the exogenous drug phorbol myristate acetate (PMA). The cells resulting from these treatments differed in term of CD4 expression. We focused our attention on interleukin‐6 (IL‐6) production, which implies an activation of the cells differentiated along both pathways. In CD4+ RA/VD‐treated cells, jacalin induced a 10‐fold higher IL‐6 secretion than did lipopolysaccharide (LPS). This jacalin‐induced IL‐6 production was inhibited by agents interacting with CD4 (anti‐CD4 mAbs and HIV recombinant gp120) or by recombinant soluble CD4. In contrast, the CD4‐ PMA‐differentiated U937 cells did not secrete any IL‐6 upon jacalin treatment, while they demonstrated a response to LPS similar to that of the RA/VD‐differentiated cells. Together with the fact that jacalin interacts with CD4, these results provide evidence of the involvement of a CD4 dependent pathway in IL‐6 production. J. Leukoc. Biol. 55: 214–220; 1994.

Cell affinity chromatography with ligands immobilized through cleavable mercury-sulfur bonds

A new methodology for cell separation by affinity chromatography is described. We have conjugated the organomercurial mersalyl to trisacryl beads bearing primary amino groups. Thiolated ligands can be immobilized on this matrix through cleavable Hg-S bonds. Two model studies of cell separation are reported: (i) concanavalin A thiolated with N-succinimidyl-3-(2-pyridyldithio)-propionate and immobilized on mersalyl-trisacryl; mouse thymocytes bound to Con A-mersalyl-trisacryl were eluted from the support by short thiol treatment which preserved cell viability; (ii) anti-dinitrophenyl antibodies modified with S-acetyl-mercaptosuccinic anhydride and immobilized on mersalyl-trisacryl; sheep erythrocytes, previously labelled with trinitrobenzene sulfonic acid, bound to this support and were easily recovered by thiol treatment without hemolysis. This methodology should overcome difficulties frequently encountered in cell affinity chromatography. Cell support multivalent interactions or high affinity of cell-ligand bindings often require drastic elution conditions which prevent viable cell recovery.

Characterization of peanut agglutinin receptors of murine thymocytes

Murine thymocytes can be separated on the basis of their agglutinability by peanut agglutinin (PNA) into two broad subpopulations assimilated to immunoincompetent agglutinated PNA+ cells and immunocompetent nonagglutinated PNA- cells. Seven surface membrane components have been isolated by immunoprecipitation using rabbit anti-PNA IgG and Staphylococcus aureus bearing protein A, from PNA-coated radiolabeled immature cells. These components (apparent molecular weights of 180, 175, 130, 115, 65, 26, and 23 kDa) labeled by the galactose oxidase/tritiated sodium borohydride method and by 125I-iodination are glycoproteins which are PNA-receptor sites normally exposed on the surface membrane of PNA+ thymocytes. The nonagglutinated PNA- cells also possess on their surface unmasked receptors for the lectin (175-180 kDa) but in lower amounts. Neuraminidase treatment prior to galactose oxidase/tritiated sodium borohydride labeling shows that the majority of PNA receptors is present on the PNA-thymocyte surface but are masked by sialic acid residues on the terminal position of the oligosaccharidic chains.

Paradoxical production of mouse thymocyte activating factor by ouabain-treated human mononuclear cells.

At concentrations as low as 10(-7) M, the cardiotonic glycosteroid ouabain, a specific inhibitor of the membrane Na+, K+-ATPase, is known to inhibit in vitro human lymphocyte proliferation produced in mixed lymphocyte cultures or induced by various stimulating agents (PHA, Con A, PWM, soluble antigens), while mouse lymphocyte proliferation is unaffected at this concentration. Ouabain inhibits most of proliferative response parameters at all stages of the transformation. This observation prompted us to suggest that ouabain could also act through inhibition of interleukin production which is known to occur during the first hours after T-cell stimulation in the presence of monocytes. In order to check the possible influence of ouabain on interleukin production, conditioned media from stimulated human mononuclear cells, prepared in the presence or in the absence of inhibitor, were tested for their ability to promote a mouse thymocyte response to PHA. Instead of the expected inhibition, we found that ouabain, even at high concentrations (2 X 10(-6) M) enhanced the stimulatory effect and/or the production of murine thymocyte activating factor(s). Moreover conditioned media from serum-free cultures of unstimulated human mononuclear cells exposed for 24 hr to low ouabain concentrations (10(-8) to 10(-7) M) showed a high activating effect on the response of murine thymocytes to PHA. This soluble factor produced upon ouabain treatment is produced by adherent cells and appears to be functionally similar to interleukin 1.

Enhancement of cell-cell contact by a nonmitogenic lectin increases blastogenic response and IL-2 release by mitogen-stimulated mouse thymocytes.

We have examined the influence of peanut agglutinin (PNA), a lectin which agglutinates but does not stimulate mouse thymocytes, on the responsiveness of these cells to concanavalin A (Con A) or galactose oxidase stimulation. Binding low amounts of PNA on unseparated mouse thymocytes pretreated with neuraminidase highly enhances the mitogenic response and the level of interleukin 2 release in the culture medium upon Con A stimulation. We have shown that PNA present on the cell surface acts as a crosslinking agent which favors intercellular binding between accessory cells (macrophages) and thymocytes, leading through this enhanced cooperation by cell-cell contact to an enhanced blastogenic response.

Unmasking of membrane enzyme activities and the problem of subcellular localization of adenylate cyclase in pig lymph node lymphocytes

A large-scale purification of plasma membranes from pig lymph node lymphocytes is described. Centrifugation on a discontinuous sucrose density gradient was performed in a zonal rotor. Adenylate cyclase activity of untreated fractions displayed a profile different from that of plasma membrane enzymatic markers and was maximal at higher density. However, when latent adenylate cyclase was unmasked by Lubrol PX treatment, its maximum was shifted to lower density and was no longer significantly different from that of plasma membrane markers. These results are discussed in terms of cell surface topography.

Study of an alcohol dehydrogenase from Rhizopus arrhizus Fisher

Abstract The in vivo reduction of ketone I (1,2-3 H -dihydro(3,2,1- kl )pyridophenothiazine-3-one) by Rhizopus arrhizus Fisher is due to a NADPH-dependent alcohol dehydrogenase. This cytosolic enzyme displays a narrow specificity for ketone I , its pH optimum being pH 8. Partially purified alcohol dehydrogenase has a good affinity for ketone I ( K m = 68 μM ).

Characterization of a Novel 2′-5′ Oligoadenylate in Stimulated Lymphocytes

Abstract We have analysed Con A-stimulated mouse lymphocytes for the presence of 2′-5′-linked oligoadenylates using a radioimmunoassay based on a monoclonal antibody raised against adenylyl (2′-5′) adenosine (A2′pA). Time-course and Con A dose dependence were performed. We found that Con A induced, in a dose-dependant manner, the accumulation of immunoreactive material, together with the incorporation of 3H-thymidine in DNA. We showed that the immunoreactive material was constituted for the essential, by a novel 2′-5′ oligoadenylate. It was isolated and characterized as adenylyl 2′-5′adenylic acid (2′ and 3′P) according to the combination of criteria such as immunoreactivity, enzyme susceptibility, chromatographic behaviour and comparison with A2′pA3′(32P)p, A2′pA3′p acid A2′pA2′p that we have chemically synthetized. This is the first example of significant variations of a 2′-5′ oligoadenylate level in circumstances other than the antiviral mechanism of interferon.

AMP-Deaminase and Cytosolic 5-Nucleotidase Involvement in Lymphocyte Maturation

Abstract AMP-Deaminase (AMP-DA) and cytosolic 5′nucleotidase, which could be involved in the cell ability to prevent cytotoxic accumulation of deoxy-ATP by nucleotide catabolism, were measured in different mouse lymphocyte subpopulations. AMP-DA is 50-fold higher in T or B splenocytes than in immature thymocytes. Cytosolic 5′nucleotidase was found similar in all lymphocyte populations. Immature T lymphoblastoid cell lines display AMP-DA activities lower than those of mature B cell lines or peripheral blood lymphocytes.