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M L Francis

Publications and source records attributed to M L Francis.

12 recordsLinked to original sources
PubMed1999

Dynamic association of L-selectin with the lymphocyte cytoskeletal matrix.

L-selectin mediates lymphocyte extravasation into lymphoid tissues through binding to sialomucin-like receptors on the surface of high endothelial venules (HEV). This study examines the biochemical basis and regulation of interactions between L-selectin, an integral transmembrane protein, and the lymphocyte cytoskeleton. Using a detergent-based extraction procedure, constitutive associations between L-selectin and the insoluble cytoskeletal matrix could not be detected. However, engagement of the L-selectin lectin domain by Abs or by glycosylation-dependent cell adhesion molecule-1, an HEV-derived ligand for L-selectin, rapidly triggered redistribution of L-selectin to the detergent-insoluble cytoskeleton. L-selectin attachment to the cytoskeleton was not prevented by inhibitors of actin/microtubule polymerization (cytochalasin B, colchicine, or nocodozole) or serine/threonine and tyrosine kinase activity (staurosporine, calphostin C, or genistein), although L-selectin-mediated adhesion of human PBL was markedly suppressed by these agents. Exposure of human PBL or murine pre-B transfectants expressing full-length human L-selectin to fever-range hyperthermia also markedly increased L-selectin association with the cytoskeleton, directly correlating with enhanced L-selectin-mediated adhesion. In contrast, a deletion mutant of L-selectin lacking the COOH-terminal 11 amino acids failed to associate with the cytoskeletal matrix in response to Ab cross-linking or hyperthermia stimulation and did not support adhesion to HEV. These studies, when taken together with the previously demonstrated interaction between the L-selectin cytoplasmic domain and the cytoskeletal linker protein alpha-actinin, strongly implicate the actin-based cytoskeleton in dynamically controlling L-selectin adhesion.

Antibodies, Monoclonal↗
PubMed1996

Loss ability to produce IFN-alpha in response to HIV-1 as monocytes differentiate into macrophages. Induction through a mechanism independent of double-stranded RNA.

IFN-alpha is an antiviral cytokine detected in plasma of HIV-1-infected patients during acute viremia and during late-stage disease. Monocytes produced IFN-alpha in response to HIV-1:1) IFN-alpha was produced predominantly by adherent cells; 2) depleting CD14+ cells nearly abolished HIV-1-induced IFN-alpha production; and 3) intracytoplasmic IFN-alpha was detected in CD14+ cells. During cell culture, monocytes differentiated into macrophages and lost their ability to produce IFN-alpha when challenged with HIV-1. These cells remained capable of producing IFN-alpha in response to other stimuli such as poly(I:C), a synthetic dsRNA. Thus, we examined two negative-stranded RNA viruses that have dsRNA intermediates, Newcastle disease virus and Sendai virus, and a DNA virus, herpes simplex virus type I (HSV-1). Macrophages lost their ability to produce IFN-alpha in response to HSV-1, but not to Sendai virus or to Newcastle disease virus. Thus, HIV-1 and other viruses were capable of inducing IFN-alpha through a mechanism that was independent of dsRNA. In conclusion, these data suggest that there are dsRNA-dependent and -independent mechanisms for the induction of IFN-alpha production, and that as monocytes differentiate into macrophages, they selectively lose their ability to produce IFN-alpha through the dsRNA-independent mechanism.

Cell Differentiation↗
PubMed1995

cAMP-independent effects of cholera toxin on B cell activation. III. Cholera toxin A subunit-mediated ADP-ribosylation acts synergistically with ionomycin or IL-4 to induce B cell proliferation.

To investigate whether ADP-ribosylation of proteins by cholera toxin could influence B cell activation, B cells were incubated with the A subunit of cholera toxin. Ionomycin acted synergistically to induce B cell proliferation with the A subunit of cholera toxin but not with cAMP-enhancing agents or with the B subunit of cholera toxin, suggesting that the synergistic effect of the A subunit was mediated via ADP-ribosylation and not via cAMP elevations or ganglioside GM1 binding. Indeed, inhibitors of ADP-ribosylation blocked the synergistic effect. Unlike anti-Ig, B cell proliferation stimulated by LPS or by the combination of the A subunit and ionomycin was observed in protein kinase C (PKC)-depleted B cells. However, neither the A subunit nor ionomycin enhanced B cell proliferation stimulated by low dose LPS, suggesting that the A subunit plus ionomycin stimulated an activation pathway distinct from the LPS-stimulated pathway. Additionally, unlike LPS, the A subunit plus ionomycin did not stimulate B cells in vitro to secrete Ig. IL-4 acted synergistically with the A subunit to induce B cell proliferation to the same extent as it did with anti-Ig; unlike the anti-Ig plus IL-4 synergy, however, the A subunit plus IL-4-mediated synergy persisted in PKC-depleted B cells. Taken together, our data suggest that cholera toxin A subunit-catalyzed ADP-ribosylation modifies a non-Gs protein involved in the activation of B cells, either through a novel pathway or at a point distal to the activation of PKC along the anti-Ig-stimulated pathway.

Adenosine Diphosphate Ribose↗
PubMed1995

Negative regulators may mediate some of the inhibitory effects of HIV-1 infected stromal cell layers on erythropoiesis and myelopoiesis in human bone marrow long term cultures.

This report presents results concerning the potential role of negative regulators in hematopoietic suppression observed in human immunodeficiency virus (HIV)-infected long-term cultures (LTC) of human bone marrow cells. Confluent stromal cell layers established from human bone marrow cells were exposed to HIV-1ADA, a monocytotropic strain of HIV-1. A progressive increase in the concentration of HIV-1 p24 antigen in cultures exposed to HIV-1ADA demonstrated that there was a productive infection. Cells from both noninfected and HIV-infected stromal cell layers produced factors that stimulated the proliferation of colony-forming units for granulocytes and macrophages (CFU-GM) from non-infected CD34+ cells. In contrast, when noninfected CD34+ cells were directly cocultured on intact stromal cell layers fewer CFU-GM and burst-forming units for erythroid cells (BFU-E) were detected in HIV-infected LTC than in noninfected LTC. One week after the addition of CD34+ cells, the number of CFU-GM in HIV-infected LTC in six of nine experiments was reduced compared to noninfected control LTC. In those six experiments, the number of CFU-GM was only 53 +/- 5% (SEM) of the number in noninfected LTC. The number of BFU-E in HIV-1-infected LTC was only 46 +/- 5% of the number in noninfected LTC (n = 5). There were fewer BFU-E in HIV-1-infected LTC, whether or not there was a reduced number of CFU-GM. Neutralizing antibody to tumor necrosis factor alpha (TNF-alpha) had no effect on the number of BFU-E in HIV-infected LTC. The number of BFU-E, however, was 2.1 +/- 0.2-fold greater (n = 3) in HIV-infected LTC incubated with neutralizing antibody to interferon-alpha. In HIV-infected LTC with decreased numbers of CFU-GM, the number of CFU-GM was approximately 2-fold greater after incubation of HIV-infected LTC with anti-interleukin-4 (IL-4). The effect of anti-TNF-alpha was variable, and anti-transforming growth factor-beta had no effect on the number of CFU-GM in HIV-infected LTC. After 2 weeks, the number of CFU-GM in HIV-infected LTC incubated with anti-IL-4 and anti-TNF-alpha was 2- to 4-fold greater than in untreated HIV-infected LTC. Antibody treatment did not promote an increase in the number of CFU-GM in noninfected LTC or in LTC in which CFU-GM numbers were not reduced after HIV infection.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigens, CD↗
PubMed1993

Induction of IFN-alpha by HIV-1 in monocyte-enriched PBMC requires gp120-CD4 interaction but not virus replication.

IFN-alpha is in plasma of HIV-1 infected patients during early and late-stage disease and may play a role in control of virus replication. The stimulus for IFN-alpha production, the cells that produce this cytokine, and the effectiveness of this IFN-alpha response for control of virus infection are not yet defined. Culture fluids from freshly isolated PBMC of HIV-1 seronegative donors contained high levels of IFN-alpha after exposure to 100 to 1000 infectious HIV-1 particles per culture. Levels of IFN-alpha induced by HIV-1 were directly dependent on the number of monocytes in cell preparations: No IFN-alpha was detected from T cell-enriched PBMC. In monocyte cultures, induction of IFN-alpha by HIV-1 was relatively specific: Levels of IL-1 beta, IL-6, IFN-gamma, and TNF-alpha remained at baseline. Capacity of HIV-1 virions to induce IFN-alpha was not dependent on virus replication. IFN-alpha was induced by (a) heat-inactivated HIV-1, (b) virions from 8E5 cells, a cell line that releases noninfectious HIV-1, (c) HIV-1-infected cells fixed in paraformaldehyde, and (d) T cell-tropic HIV-1 that binds to but does not infect monocytes. Capacity of HIV-1 virions and HIV-1 infected cells to induce IFN-alpha was completely inhibited by soluble rCD4 or mAb against CD4 or gp120. Antibodies against CD4, however, did not induce monocytes to produce IFN-alpha. HIV-1-induced IFN-alpha production was inhibited by antibodies against both V3 loop determinants and the CD4 binding site of gp120. Further, sera and purified immunoglobulin from HIV-1 infected patients also inhibited HIV-1-induced IFN-alpha production. These observations suggest that potentially protective antiviral responses associated with IFN-alpha production in HIV-1 infected patients are inhibited by the development of antibodies against gp120.

CD4 Antigens↗
PubMed1992

Cyclic AMP-independent effects of cholera toxin on B cell activation. II. Binding of ganglioside GM1 induces B cell activation.

Although the physiologic function of gangliosides is unknown, evidence suggests they play a role in the regulation of cell growth. The binding of ganglioside GM1 by recombinant B subunit of cholera toxin (rCT-B) inhibited mitogen-stimulated B cell proliferation without elevating intracellular cAMP. CT-B paradoxically enhanced the expression of MHC class II (Ia) molecules and minor lymphocyte-stimulating determinants without altering the expression of some other immunologically relevant B cell surface Ag. Increased expression of Ia was not detected until 4 h after stimulation, kinetics similar to those seen when B cells are stimulated with anti-Ig antibody or IL-4, suggesting that the enhancement was not the result of redistribution of existing cell surface markers but rather the result of a new metabolic event. Both the inhibitory and stimulatory effects of CT-B could be blocked by incubation of CT-B with ganglioside GM1. Furthermore, enhancement of the CT-B-mediated effect was seen when additional ganglioside GM1 was incorporated into the B cell membrane. rCT-B with a mutation that interfered with its binding to ganglioside GM1 did not enhance Ia expression. Taken together, these results indicate that the observed effects of CT-B were most likely mediated through the binding of cell surface ganglioside GM1. CT-B-mediated stimulation of Ia expression provides a potential explanation for the previously described ability of CT-B to act as an immunoadjuvant. These results suggest that the binding of ganglioside GM1 has multiple B cell growth-regulating effects.

Animals↗
PubMed1992

Interferons in the persistence, pathogenesis, and treatment of HIV infection.

Interferon (IFN) plays an important role in the treatment and pathogenesis of HIV disease. Recent studies show beneficial effects of IFN alpha in the treatment of HIV-associated Kaposi's Sarcoma and early HIV-infection. Moreover, cell culture studies support these beneficial effects. HIV infection of monocytes is blocked by IFN alpha administered at the time of viral challenge. The IFN alpha-treated cells show no evidence of HIV infection. Viral gene products produced in monocytes infected with HIV then treated with IFN alpha gradually decrease to baseline. Large quantities of proviral DNA are seen in the HIV-infected IFN alpha-treated cells with little evidence for viral transcription suggesting true microbiological latency. While most viral infections of cells result in IFN production, HIV is a notable exception. Indeed, HIV does not induce monocytes to produce IFN alpha and blocks its production following poly(I).poly(c) stimulation. This allows HIV yet another mechanism to evade an important host antiviral response. Paradoxically, the appearance of IFN activity in sera of HIV-infected patients is associated with disease progression, not resolution. Recent observations showing that the interaction between HIV-infected monocytes and PBMC results in the production of IFN alpha s with reduced anti-HIV activity may help explain this paradox. Thus, IFN alpha plays an important but complex role in HIV disease. The elucidation of cellular factors that regulate the antiretroviral effects of IFN alpha may lead to the development of novel therapeutic strategies for HIV infection.

Animals↗
PubMed1991

Identification of a prominent 85-kDa cAMP-dependent phosphoprotein associated with late G1 phase in mitogen-stimulated B lymphocytes.

In order to elucidate late regulatory events which may be involved in the onset of S phase in B lymphocytes, we studied the effect of anti-Ig on phosphorylation of soluble proteins at late G1 phase. Stimulation of murine splenic B lymphocytes with anti-Ig and other mitogens for 18 h was found to be associated with a major increase in phosphorylation of an 85 kDa/pI approximately 5.3 cytosolic protein, conversely, stimulation of the cells with non-mitogenic stimuli did not induce the phosphorylation of pp85. The increase in phosphorylation of pp85 could not be detected after 30 min, was barely detectable after 6 h, but was very prominent after 18 h of stimulation with anti-Ig. Thus, the increase in phosphorylation of pp85 is not an early signal but is rather correlated with the late G1 phase. pp85 could not be detected in the nuclei of either control or stimulated cells. Stimulation of B cells for 30 min with forskolin induced the phosphorylation of pp85, while phorbol ester did not have any effect. The phosphorylation of pp85 was induced by the catalytic subunit of cAMP protein kinase. Comparison of the phosphopeptide map of pp85 phosphorylated by anti-Ig in intact cells to the phosphopeptide map phosphorylated by forskolin or by the catalytic subunit of cAMP protein kinase, showed a striking similarity indicating that cAMP protein kinase may be involved in phosphorylation of pp85 in mitogen-stimulated cells. An increase in intracellular cAMP levels at late G1 phase was found in B cells stimulated by mitogens. These results implicate an important role for cAMP-dependent phosphorylation events, specifically the phosphorylation of pp85/pI 5.3, at late G1 phase during the cell cycle.

Animals↗
PubMed1990

cAMP-independent effects of cholera toxin on B cell activation. I. A possible role for cell surface ganglioside GM1 in B cell activation.

Cholera toxin has been used as a tool to study the effects of cAMP on the activation of B cells but may have effects independent of its ability to elevate cAMP. We found five lines of evidence which suggested that cholera toxin suppressed mitogen-stimulated B cell activation through a cAMP-independent pathway. 1) Cholera toxin (1 microgram/ml) was consistently more suppressive than forskolin (100 microM) despite the induction of higher intracellular cAMP levels by forskolin. 2) Cholera toxin was more suppressive at 1 microgram/ml than at 0.1 microgram/ml despite equivalent elevations of cAMP. 3) Washing B cells following their incubation with cholera toxin reversed much of the inhibition without altering intracellular cAMP levels. 4) The A subunit of cholera toxin, which at high concentrations (10 micrograms/ml) induced levels of cAMP comparable to those induced by cholera toxin (1 and 0.1 microgram/ml), did not inhibit B cell activation. 5) cAMP derivatives at high concentrations were much less effective than was cholera toxin in suppressing B cell activation. Although the elevation of cAMP may cause a mild inhibition of B cell proliferation, we found that even a marked elevation of cAMP did not suppress B cell proliferation, unless the elevation was persistent. We did, however, observe that the degree of toxin inhibition more closely paralleled binding of the toxin to B cells than toxin stimulation of cAMP. This result raised the possibility that binding of cholera toxin to its ganglioside GM1 receptor mediated an inhibitory signal which suppressed B cell proliferation.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1990

Effects of cholera toxin on human B cells. Cholera toxin induces B cell surface DR expression while it inhibits anti-mu antibody-induced cell proliferation.

Experiments were performed to investigate the effect of cholera toxin (CT) on human B cell function. Highly purified (greater than 98% CD20+) human peripheral blood B cells were exposed to CT in the presence or absence of anti-mu antibody. Treatment of highly purified B cells with CT stimulated enhanced expression of surface DR molecules, whereas it did not enhance expression of other B cell surface activation markers including transferrin or IL-2R. Neither the A nor the B subunits of CT by themselves enhanced the expression of surface DR Ag. In addition, 8-bromo-cAMP alone or in combination with the B subunit did not increase the expression of human B cell surface DR Ag. These findings suggest that neither elevation of cAMP nor binding to GM1 ganglioside are sufficient to stimulate this activation parameter in B cells. Associated with CT-mediated enhanced expression of MHC class II molecules we found that CT-treated B cells also served as stronger stimulators, compared with control cells, of both autologous and allogeneic MLR responses in peripheral blood T cells. Although CT stimulated early events in B cell activation, it inhibited anti-mu antibody-induced B cell thymidine incorporation by 55 to 75%. Inhibitory effects of CT were observed even when CT was added to cultures as late as 36 h after the addition of the anti-mu antibody. These results suggest that CT has both a stimulatory and inhibitory effect on human B cells and that the stimulatory effect may be mediated via a cAMP-independent mechanism.

8-Bromo Cyclic Adenosine Monophosphate↗