ABSTRACT
A nonpeptidyl small molecule SB 247464, capable of activating granulocyte-colony-stimulating factor (G-CSF) signal transduction pathways, was identified in a high-throughput assay in cultured cells. Like G-CSF, SB 247464 induced tyrosine phosphorylation of multiple signaling proteins and stimulated primary murine bone marrow cells to form granulocytic colonies in vitro. It also elevated peripheral blood neutrophil counts in mice. The extracellular domain of the murine G-CSF receptor was required for the activity of SB 247464, suggesting that the compound acts by oligomerizing receptor chains. The results indicate that a small molecule can activate a receptor that normally binds a relatively large protein ligand.
Subject(s)
Benzimidazoles/pharmacology , Guanidines/pharmacology , Milk Proteins , Proto-Oncogene Proteins , Receptors, Granulocyte Colony-Stimulating Factor/metabolism , Animals , Benzimidazoles/chemistry , Benzimidazoles/metabolism , Cell Line , Colony-Forming Units Assay , DNA-Binding Proteins/metabolism , Dimerization , Female , Granulocyte Colony-Stimulating Factor/metabolism , Granulocyte Colony-Stimulating Factor/pharmacology , Granulocytes/cytology , Guanidines/chemistry , Guanidines/metabolism , Humans , Janus Kinase 1 , Janus Kinase 2 , Leukocyte Count , Leukopoiesis , Mice , Mice, Inbred C57BL , Neutrophils/cytology , Phosphorylation , Phosphotyrosine/metabolism , Protein-Tyrosine Kinases/metabolism , Receptors, Granulocyte Colony-Stimulating Factor/chemistry , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , STAT3 Transcription Factor , STAT5 Transcription Factor , Signal Transduction/drug effects , Species Specificity , Trans-Activators/metabolism , Transfection , Tumor Cells, CulturedABSTRACT
Blood monocytes from patients with active tuberculosis are activated in vivo, as evidenced by an increase in the stimulated release of proinflammatory cytokines, such as TNF-alpha, and the spontaneous expression of IL-2R. Further, monocytes from patients demonstrate an augmented susceptibility to a productive infection with HIV-1 in vitro. Mycobacterium tuberculosis and its components are strong signals to activate monocytes to production of cytokines. In this study we examined the basis of activation of monocytes during active tuberculosis and by M. tuberculosis. We found a constitutive degradation of I kappa B-alpha, the major cytoplasmic inhibitor of nuclear factor kappa B (NF-kappa B), in freshly isolated PBMC and monocytes from patients with tuberculosis. In contrast, I kappa B-alpha levels in PBMC and monocytes from healthy subjects or from patients with nontuberculous pulmonary conditions were intact. Further, by electrophoretic mobility shift assay, NF-kappa B was activated in monocytes from tuberculous patients. The expression of I kappa B-alpha gene, which is responsive to activation by NF-kappa B, was up-regulated in PBMC and monocytes from patients, but not in mononuclear cells from healthy subjects or those with nontuberculous lung diseases. By contrast, the expression of other adherence-associated early genes, such as IL-8 and IL-1 beta, was not up-regulated in PBMC of tuberculous patients. Further, M. tuberculosis and its tuberculin, purified protein derivative, induced the degradation of I kappa B-alpha and the expression of I kappa B-alpha mRNA, and purified protein derivative induced the activation of NF-kappa B in monocytes.
Subject(s)
DNA-Binding Proteins/metabolism , I-kappa B Proteins , Monocytes/metabolism , Monocytes/microbiology , Mycobacterium tuberculosis/immunology , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Tuberculosis/immunology , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Gene Expression Regulation/immunology , Humans , NF-KappaB Inhibitor alpha , RNA, Messenger/biosynthesis , Tuberculin/pharmacology , Tuberculosis/metabolismABSTRACT
Native 30-kD antigen, also known as alpha antigen, is a fibronectin-binding protein that is secreted by live Mycobacterium tuberculosis. This antigen may play an important biological role in the host-parasite interaction since it elicits delayed type hypersensitivity response and protective immunity in vivo and T lymphocyte blastogenesis and IFN-gamma production in vitro. In the present study, we show that, TNF-alpha protein is produced in monocyte culture supernatants in response to 30-kD antigen and the level is as high as that to purified protein derivative of M. tuberculosis. This stimulatory effect was not due to contamination with either bacterial lipopolysaccharide or mycobacterial lipoarabinomannan. The preincubation of monocytes with plasma fibronectin significantly enhanced the release of TNF-alpha into the culture supernatants in response to 30-kD antigen. This effect was blocked by polygonal antibody to plasma fibronectin. In contrast, the monocytic cell line U937 failed to release TNF-alpha protein in the culture supernatants in response to 30-kD antigen with or without preincubation with plasma fibronectin. To determine whether this observation was due to differential binding of the 30-kD to fibronectin on these cells, a cell based ELISA was used. Pretreatment of monocytes with fibronectin enhanced their binding of the 30-kD antigen. U937 cells bound the 30-kD antigen weakly with or without fibronectin pretreatment. These results indicate that 30-kD antigen which is a known secretary antigen of M. tuberculosis is a stimulus for human monocytes to express TNF-alpha and that stimulatory effect may be mediated through plasma fibronectin.
Subject(s)
Antigens, Bacterial/pharmacology , Fibronectins/pharmacology , Monocytes/drug effects , Tumor Necrosis Factor-alpha/biosynthesis , Antigens, Bacterial/metabolism , Drug Synergism , Enzyme-Linked Immunosorbent Assay , Fibronectins/metabolism , Humans , Protein BindingABSTRACT
The 30-kDa secreted antigen of Mycobacterium tuberculosis was a strong inducer of tumor necrosis factor alpha in human monocytes. Our findings suggest that tumor necrosis factor alpha production may be up-regulated at both the posttranscriptional and transcriptional levels. Regulation at the posttranscriptional level probably reflects enhanced translational efficiency.
Subject(s)
Antigens, Bacterial/immunology , Monocytes/immunology , Mycobacterium tuberculosis/immunology , Tumor Necrosis Factor-alpha/biosynthesis , Antigens, Bacterial/chemistry , Humans , In Vitro Techniques , Molecular Weight , RNA, Messenger/metabolism , Tumor Necrosis Factor-alpha/geneticsABSTRACT
These studies were undertaken to examine the in vitro effects of the cysteine pro-drug L-2-oxothiazolidine-4-carboxylic acid (Procysteine) on human immunodeficiency virus expression. Procysteine inhibited HIV expression in human peripheral blood mononuclear cells as detected by measurement of supernatant core antigen. In transient transfection assays, Procysteine inhibited gene expression controlled by the HIV-1 promoter in activated Jurkat cells but not in resting Jurkat cells. Gel-shift assays showed that Procysteine inhibited NF-kappa B DNA binding activity in nuclear extracts. Procysteine did not affect the production of interleukin-2 by peripheral blood mononuclear cells of healthy HIV-seronegative subjects, as measured by bioassay but it decreased the density of cell-surface interleukin-2 receptors detected by flow cytometry after stimulation with phytohemagglutinin (PHA). Thus, Procysteine inhibits HIV expression, HIV promoter activity, and NF-kappa B binding activity in vitro. Procysteine does not affect interleukin-2 production but inhibits interleukin-2 receptor expression in PHA-stimulated peripheral blood mononuclear cells.
Subject(s)
HIV Infections/virology , HIV-1/drug effects , Leukocytes, Mononuclear/virology , Receptors, Interleukin-2/drug effects , Thiazoles/pharmacology , Cells, Cultured , Dose-Response Relationship, Drug , Gene Expression/drug effects , HIV Core Protein p24/analysis , HIV-1/genetics , HIV-1/growth & development , Humans , Interleukin-2/pharmacology , Lymphocyte Activation/drug effects , NF-kappa B/metabolism , Phytohemagglutinins/pharmacology , Promoter Regions, Genetic , Protein Binding , Pyrrolidonecarboxylic Acid , Receptors, Interleukin-2/biosynthesis , T-Lymphocytes/virology , Thiazolidines , Virus Replication/drug effectsABSTRACT
We examined the ability of purified protein derivative (PPD) of Mycobacterium tuberculosis to induce transforming growth factor beta 1 (TGF-beta 1), a potent immunosuppressive and macrophage-deactivating molecule, in blood monocytes from healthy individuals. TBF-beta 1 activity in PPD-induced monocyte supernatants was identified by Western immunoblot analysis and was not inhibited by polymyxin B, an inhibitor of bacterial lipopolysaccharide (LPS). Furthermore, PPD at equivalent amounts in weight to LPS was as potent in stimulation of monocyte production of TGF-beta 1 at 24 h of culture, as quantified by enzyme-linked immunosorbent assay. The inducing effect of PPD, in contrast to that of LPS, was sustained at later time points of culture (72 h). PPD enhanced the constitutive expression of TGF-beta 1 steady-state mRNA in monocytes at 24 and 48 h of culture. In contrast, neither mycobacterial heat shock protein (64-kDa protein of M.bovis) nor LPS induced TGF-beta 1 mRNA. Decay studies suggested a transcriptional rather than a posttranscriptional effect of PPD on TGF-beta 1 gene expression.
Subject(s)
Monocytes/drug effects , Mycobacterium tuberculosis/immunology , Transforming Growth Factor beta/biosynthesis , Tuberculin/pharmacology , Blotting, Western , Humans , Lipopolysaccharides/pharmacology , Polymyxin B/pharmacology , RNA, Messenger/metabolism , Transcription, Genetic , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/isolation & purificationABSTRACT
The production of transforming growth factor-beta (TGF beta 1) by human monocytes (MN) infected with Mycobacterium tuberculosis and its effects on the intracellular fate of the organism were studied. M. tuberculosis infection of MN induced both expression of mRNA and secretion of tumor necrosis factor-alpha (TNF alpha) and TGF beta 1 protein. Neutralizing antibody to TGF beta 1 reduced the intracellular growth of M. tuberculosis. The growth-enhancing effects of TGF beta 1 could not be explained by increased initial bacterial load. Preculture with TGF beta 1 decreased uptake of M. tuberculosis. Exposure of MN to increasing concentrations of TGF beta 1 before or after infection with M. tuberculosis accelerated intracellular bacterial replication. Both TNF alpha and interferon-gamma (IFN-gamma) limited mycobacterial replication. TGF beta 1 (10 ng/mL) abrogated the bacteriostatic effects of TNF alpha and IFN-gamma. Within the infected focus, TGF beta 1 produced by mononuclear phagocytes may play an important role in the pathogenesis of tuberculosis, in part by modulating the response to potentially protective cytokines such as TNF alpha and IFN-gamma.
Subject(s)
Monocytes/microbiology , Mycobacterium tuberculosis/drug effects , Transforming Growth Factor beta/pharmacology , Humans , Interferon-gamma/pharmacology , Monocytes/metabolism , Mycobacterium tuberculosis/growth & development , Phagocytosis/drug effects , Transforming Growth Factor beta/biosynthesis , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/pharmacology , Virus Replication/drug effectsABSTRACT
We have developed a novel method for screening a Mycobacterium bovis (BCG) cosmid library in Mycobacterium smegmatis for the detection of immunostimulatory T-cell antigens (Ag). Distinctive protein banding patterns were demonstrated in culture filtrates of three of 30 recombinant M. smegmatis clones: pBCCS13 (41 and 73 kDa); pBCCS221 (30, 50 and 68 kDa); pBCCS223 (100 kDa). Western immunoblots indicated that monoclonal antibodies (mAb) directed to the previously characterized 19-, 30-, 38-, 65- and 71-kDa mycobacterial Ag were not reactive with the distinctive recombinant proteins. Furthermore, T-cell Western blots demonstrated that fractions containing the distinctive proteins were immunostimulatory. A given tuberculin-positive donor expressed unique patterns of blastogenic reactivity to protein fractions isolated from each of the three recombinant clones. Restriction enzyme digests of the three recombinant BCG inserts revealed distinctive DNA-banding patterns. The immunostimulatory Ag, therefore, are most likely encoded within different regions of the BCG genome, as contained within three distinct inserts. T-cell Western blots further indicated a heterogeneity in the repertoire of BCG-responsive T cells since tuberculin-positive donors varied in the pattern of reactivity to protein fractions isolated from the same recombinant filtrate. Most likely, immunity to M. tuberculosis results from activation of a heterogeneous array of T cells targeted to multiple immunostimulatory Ag. The method we describe should greatly enhance our ability to define the full spectrum of T-cell Ag encoded by mycobacteria, particularly those which are secreted proteins.
Subject(s)
Antigens, Bacterial/immunology , Mycobacterium bovis/immunology , Mycobacterium/immunology , T-Lymphocytes/immunology , BCG Vaccine/immunology , Blotting, Western , Electrophoresis, Polyacrylamide Gel , In Vitro Techniques , Lymphocyte Activation , Recombinant Proteins/immunologyABSTRACT
This study demonstrated for the first time that bone marrow is a target of enhanced in vivo monocytopoiesis in hyperlipemia. A significantly greater number of bone marrow cells (BMC) were recovered per femur in swine fed a hyperlipemic (HL) diet compared with swine fed a normal (N) diet. In addition, a significantly elevated number of monocytic precursors proliferated in HL-swine compared with N-swine BMC cultures grown in standardized media in the absence of an exogenous source of colony stimulating factor (CSF). HL-swine sera stimulated a significant enhancement in the number of proliferating monocytic precursor cells, regardless of whether or not the BMC were from HL- or N-swine. In addition, HL-swine compared with N-swine BMC demonstrated an enhanced intrinsic capacity to form monocytic colonies in culture, irrespective of the source of swine sera used to stimulate growth.
Subject(s)
Bone Marrow/pathology , Hematopoietic Stem Cells/pathology , Hyperlipidemias/pathology , Monocytes/pathology , Animals , Bone Marrow/enzymology , Cell Division , Colony-Stimulating Factors/metabolism , Esterases/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor , Growth Substances/metabolism , Hematopoietic Stem Cells/enzymology , Histocytochemistry , Hyperlipidemias/enzymology , Lymphocytes/enzymology , Lymphocytes/pathology , Male , Monocytes/enzymology , Neutrophils/enzymology , Neutrophils/pathology , SwineABSTRACT
Cyclic AMP has long been proposed to be the intracellular second messenger that conveys the inhibitory signal for T-cell activation and clonal T-cell proliferation. The present study further explores the mechanism by which the cAMP pathway regulates human T-lymphocyte interleukin-2 (IL-2) production and T-cell blastogenesis. Activation of adenylate cyclase, inhibition of cAMP-dependent phosphodiesterase, or the direct addition of the cell-permeable cAMP analog, 8-N3-cAMP, increased occupancy of intracellular cAMP receptors, inhibited IL-2 production, and reduced T-cell proliferation. However, inhibition of cAMP-dependent protein phosphorylation by N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), a cell-permeable inhibitor of cyclic nucleotide-dependent protein kinase, partially restored IL-2 production. Our data support the conclusion that the cAMP pathway conveys an inhibitory signal for IL-2 production and T-cell proliferation via an integral protein phosphorylation step.
Subject(s)
Cyclic AMP/physiology , Interleukin-2/biosynthesis , T-Lymphocytes/metabolism , 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone/pharmacology , Adenylyl Cyclases/metabolism , Adolescent , Adult , Aged , Azides/pharmacology , Calcimycin/pharmacology , Cell Line , Colforsin/pharmacology , Cyclic AMP/analogs & derivatives , Cyclic AMP/pharmacology , Dose-Response Relationship, Immunologic , Female , Humans , Immunosuppressive Agents/pharmacology , Interleukin-1/pharmacology , Interleukin-2/antagonists & inhibitors , Lymphocyte Activation/drug effects , Male , Middle Aged , Phosphorylation , T-Lymphocytes/drug effects , T-Lymphocytes/enzymology , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
An analysis was made of the ability of bone marrow cells derived from young (6 months) or old (28 months) murine (A X C57BL/6) donors to differentiate to B and T lymphocytes following serial bone marrow cell transplantations into groups of young syngeneic lethally irradiated recipients. Both number and mitogenic responsiveness of splenic B and T lymphocytes were recorded for recipients of young or old bone marrow cells 9 months following each serial transplant. The data suggests that lymphopoietic stem cells senescence in a manner analogous to that predicted by the clonal succession model of ageing.
Subject(s)
B-Lymphocytes/immunology , Bone Marrow/growth & development , Lymphocyte Activation , T-Lymphocytes/immunology , Aging , Animals , Bone Marrow Transplantation , Female , Lipopolysaccharides , Mice , Mice, Inbred Strains , Phytohemagglutinins , Spleen/immunologyABSTRACT
Changes in splenic B and T lymphocyte number and mitogenic activity with age were quantitated in (A X C57BL/6)F1 (AB6F1) hybrid mice. Although both the B and T lymphocyte proliferative reactivity to their respective mitogens, lipopolysaccharide (LPS) and phytohemagglutinin (PHA), declined significantly with age, an earlier and more marked reduction was recorded for the T cell response. The decline in B and T lymphocyte mitogenic activity with age could not be correlated with a corresponding reduction in the percentage of splenic B or T lymphocytes. The main focus of this study was to determine if the reduction in T and B lymphocyte mitogenic activity with age results primarily from a mechanism intrinsic to the lymphoid lineage itself or from adverse extracellular factors that increase with age. Bone marrow cells (BMC) derived from individual young and old donor AB6F1 mice were transplanted into the neutral environment of young, lethally irradiated syngeneic recipients. Number and mitogenic activity of splenic T and B lymphocytes were recorded for the original BMC donors as well as for the recipients of the young and old BMC lines 9 mo after the BMC transplants. A predominance of the donor (male) rather than recipient (female) karyotype within the mitogen-responding populations of recipient mice confirmed a donor BMC take. The PHA and LPS response levels exhibited by the old donors were 30% and 70% of those of the young donors, respectively. These differences in PHA and LPS reactivity recorded between young and old donors were maintained between recipients of young and old donor BMC lines. Thus, even under the influence of a young recipient environment, old BMC were incapable of giving rise to mitogen responding cells with a functional competence equivalent to that of their younger counterparts. This finding would lend further support to the theory that an intrinsic mechanism is responsible for the decline in murine mitogenic activity with age.
Subject(s)
Aging , B-Lymphocytes/immunology , Lymphocyte Activation , Spleen/cytology , T-Lymphocytes/immunology , Animals , B-Lymphocytes/physiology , Cell Differentiation , Crosses, Genetic , Female , Leukocyte Count , Lipopolysaccharides/pharmacology , Male , Mice , Phytohemagglutinins/pharmacology , Spleen/immunology , Spleen/physiology , T-Lymphocytes/physiologyABSTRACT
The effects of serial transplantation on murine bone marrow were studied. Decline of CFUs content and the maximum number of successful transplantations possible were the criteria considered to indicate stem cell changes accompanying the serial transplants. Variables of experimentation included age of the marrow cell donors, addition of thymocytes to the transplanted marrow, differing time intervals between transplantations and differing numbers of donor marrow cells injected throughout a given transplant series. All of the transplantation series were monitored by karyotyping the marrow cells after each transplantation to document the continuance of the original donor cells. Reversions to recipient cell type did occur late in some series and were considered the end point of these series, as were the disappearance of CFUs and deaths of recipients due to transplant failure (the latter two always corresponded well). Only the donor marrow cell dose was found to be an important variable in the continuance of a transplant series and it was possible to obtain 8 successful serial transplantations with the highest marrow cell dose. All of the series given lower cell doses failed at the fifth or sixth transplant, regardless of other variables involved. A naturally occurring hierarchy of hemopoietic stem cells coupled with stem cell differentiative changes caused by extreme and repeated demands for population replenishment is offered as a probable reason for the results obtained, while other possibilities are discussed as well.