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2.
Oncogene ; 29(28): 4113-20, 2010 Jul 15.
Article in English | MEDLINE | ID: mdl-20453890

ABSTRACT

The Myc-deregulating chromosomal T(12;15)(Igh-Myc) translocation, the hallmark mutation of inflammation- and interleukin 6-dependent mouse plasmacytoma (PCT), is the premier model of cancer-associated chromosomal translocations because it is the only translocation in mice that occurs spontaneously (B lymphocyte lineage) and with predictably high incidence (approximately 85% of PCT), and has a direct counterpart in humans: Burkitt lymphoma t(8;14)(q24;q32) translocation. Here, we report on the development of a genetic system for the detection of T(12;15)(Igh-Myc) translocations in plasma cells of a mouse strain in which an enhanced green fluorescent protein (GFP)-encoding reporter gene has been targeted to Myc. Four of the PCTs that developed in the newly generated translocation reporter mice, designated iGFP(5'Myc), expressed GFP consequent to naturally occurring T(12;15) translocation. GFP expression did not interfere with tumor development or the deregulation of Myc on derivative 12 of translocation, der (12), because the reporter gene was allocated to the reciprocal product of translocation, der (15). Although the described reporter gene approach requires refinement before T(12;15) translocations can be quantitatively detected in vivo, including in B lymphocyte lineage cells that have not yet completed malignant transformation, our findings provide proof of principle that reporter gene tagging of oncogenes in gene-targeted mice can be used to elucidate unresolved questions on the occurrence, distribution and trafficking of cells that have acquired cancer-causing chromosomal translocations of great relevance for humans.


Subject(s)
Genes, Reporter , Genes, myc , Immunoglobulin Heavy Chains/genetics , Oncogenes , Translocation, Genetic , Animals , Green Fluorescent Proteins/genetics , In Situ Hybridization, Fluorescence , Mice , Polymerase Chain Reaction
3.
Am J Transplant ; 9(10): 2270-80, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19681828

ABSTRACT

Mast cells (MC) have been shown to mediate regulatory T-cell (T(reg))-dependent, peripheral allograft tolerance in both skin and cardiac transplants. Furthermore, T(reg) have been implicated in mitigating IgE-mediated MC degranulation, establishing a dynamic, reciprocal relationship between MC and T(reg) in controlling inflammation. In an allograft tolerance model, it is now shown that intragraft or systemic MC degranulation results in the transient loss of T(reg) suppressor activities with the acute, T-cell dependent rejection of established, tolerant allografts. Upon degranulation, MC mediators can be found in the skin, T(reg) rapidly leave the graft, MC accumulate in the regional lymph node and the T(reg) are impaired in the expression of suppressor molecules. Such a dramatic reversal of T(reg) function and tissue distribution by MC degranulation underscores how allergy may causes the transient breakdown of peripheral tolerance and episodes of acute T-cell inflammation.


Subject(s)
Cell Degranulation , Immune Tolerance , Mast Cells/pathology , Animals , Base Sequence , DNA Primers , Graft Rejection , Heart Transplantation/immunology , Inflammation Mediators/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Skin Transplantation/immunology , T-Lymphocytes/immunology , Transplantation, Homologous
4.
Alcohol ; 24(3): 155-67, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11557301

ABSTRACT

Patients with chronic inflammatory diseases, including Crohn's disease and rheumatoid arthritis, as well as those with certain viral infections, and patients who are transplant recipients or who have certain hematologic malignancies have been observed to have CD57+ T cell expansion in both CD4+ and CD8+ subsets. We have reported previously that alcoholic patients also have CD57+ T cell expansion. Because many alcoholics become seriously deficient in cell-mediated immunity, it is of interest to determine whether the expanded CD57+ subsets can respond to stimulation with normal T helper cell subtype 1 (TH1) cytokine production. We report evaluation of the CD57 T-cell subsets of patients with alcoholic liver disease (ALD) with the use of cytoplasmic staining after stimulation through the T-cell receptor (TCR). The CD57+ subsets of the T cells of both healthy individuals and patients with ALD express significantly higher amounts of cytoplasmic tumor necrosis factor-alpha (TNF-) and interferon-gamma (IFN-) after 6 h of stimulation than do the CD57- subsets. This increased production can persist up to 46 h of continuous stimulation. Under these assay conditions, very little cytoplasmic interleukin (IL)-4 is observed in the T cells of either healthy control subjects or patients with ALD. Measurement of cytokine secretion by sort-purified CD57 T-cell subsets with the use of enzyme-linked immunosorbent assay (ELISA) shows that the CD57+ T-cell subset produces 18- to 30-fold more TNF- and IFN-, respectively, than does the CD57- subset in the first 12 h of stimulation. This response requires only stimulation through the TCR for the CD57+ subset, whereas significant secretion by the CD57- subset requires added IL-2 or anti-CD28 antibody. These results are consistent with the concept of the CD57+ T-cell subset as a differentiated effector cell and demonstrate that patients with ALD who are not drinking at the time of evaluation have normal or increased immediate TH1 T-cell responses.


Subject(s)
CD57 Antigens/biosynthesis , Cytokines/biosynthesis , Liver Diseases, Alcoholic/immunology , T-Lymphocyte Subsets/metabolism , Th1 Cells/metabolism , Adult , Cytoplasm/immunology , Cytoplasm/metabolism , Humans , Immunophenotyping , Interferon-gamma/biosynthesis , Interleukin-4/biosynthesis , Linear Models , Liver Diseases, Alcoholic/metabolism , Middle Aged , T-Lymphocyte Subsets/immunology , Th1 Cells/immunology , Tumor Necrosis Factor-alpha/biosynthesis
5.
Science ; 293(5537): 2012-3, 2001 Sep 14.
Article in English | MEDLINE | ID: mdl-11557866

ABSTRACT

What determines whether transitional B cells newly emerged from the bone marrow will differentiate further to become mature, long-lived, circulating B lymphocytes? In a Perspective, Waldschmidt and Noelle discuss new findings showing that the TNF family ligand BAFF and its receptor BAFF-R are crucial for selecting transitional B cells into the mature B cell pool (Thompson et al., Schiemann et al.).


Subject(s)
B-Lymphocytes/immunology , B-Lymphocytes/physiology , Membrane Proteins/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Tumor Necrosis Factor-alpha/metabolism , Animals , B-Cell Activating Factor , B-Cell Activation Factor Receptor , B-Cell Maturation Antigen , B-Lymphocytes/metabolism , Bone Marrow Cells , Cell Survival , Immunoglobulin M/biosynthesis , Ligands , Mice , Mice, Inbred A , Mice, Knockout , Mice, Mutant Strains , Receptors, Tumor Necrosis Factor/genetics , Signal Transduction , Spleen/cytology , Spleen/immunology , Transmembrane Activator and CAML Interactor Protein
6.
Arch Environ Contam Toxicol ; 40(3): 311-7, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11443360

ABSTRACT

Soils from the Yorktown Naval Base contaminated with trinitrotoluene (TNT) and other explosives were used to prepare eluates before and after bioremediation using microbial growth amendments in the presence (P1 eluates) or absence (P2 eluates) of exogenous white rot fungus. Effectiveness of bioremediation was examined by several immunotoxicity assays-viability/growth of lymphocytes, cytokine production, and expression of the interleukin-2 (IL-2) receptor-using human peripheral blood mononuclear cells exposed to the eluates. Although TNT concentrations decreased in both P1 and P2 eluates relative to untreated baseline soil (BL) eluates, a recovery in lymphocyte growth/viability and IL-2 secretion was seen with P2 but not P1 eluates relative to BL eluates. IL-2 receptor levels were higher in cells exposed to BL and P2 eluates than when exposed to P1 eluates. Interferon-gamma, tumor necrosis factor-beta, and IL-10 levels were highest in BL and P2 eluates and lowest in P1 eluates. Taken together, these results suggest that treatment of the soil with microbial growth amendments in the absence but not the presence of exogenous white rot fungi lead to partial bioremediation as assessed by lymphocyte functions.


Subject(s)
Soil Pollutants/toxicity , Trinitrotoluene/toxicity , Biodegradation, Environmental , Fungi , Humans , Immunotoxins/metabolism , Immunotoxins/toxicity , Lymphocytes/drug effects , Lymphocytes/physiology , Soil Microbiology , Soil Pollutants/metabolism , Trinitrotoluene/metabolism
7.
Cytometry ; 43(4): 297-307, 2001 Apr 01.
Article in English | MEDLINE | ID: mdl-11260597

ABSTRACT

BACKGROUND: Many methods have been developed specifically for identifying hematopoietic progenitor cells in murine bone marrow, but few methods allow rapid identification of multiple bone marrow populations. We describe a new, simple method for identifying simultaneously eight populations in murine bone marrow with two-color flow cytometry and phenotypically define these populations. METHODS: Bone marrow was stained with anti-Ly-6C and anti-B220 (CD45R) in one fluorochrome and wheat germ agglutinin (WGA) in another fluorochrome. The eight populations identified in this way were defined further primarily by four-color flow cytometry. RESULTS: Six of the eight populations were characterized phenotypically as containing erythroid, granulocytic, mast, early B, mature B, and stem cell populations. Two additional populations with phenotypic characteristics of partially differentiated precursor cells also were identified. One population was Ly-6C/B220+ and WGA-. It also expressed markers associated with early B, T, and/or dendritic cell differentiation. The second population was Ly-6C(hi)WGA(hi)Mac-1+ and was negative for numerous other lineage-specific and precursor markers. Its morphology suggested monocytic differentiative potential. CONCLUSIONS: A two-color flow cytometric assay profiles six bone marrow populations with identifiable phenotypes and two additional unique, putative hematopoietic precursor populations.


Subject(s)
Bone Marrow Cells/cytology , Flow Cytometry/methods , Hematopoietic Stem Cells/cytology , Animals , Antigens, Ly/analysis , Bone Marrow Cells/chemistry , Bone Marrow Cells/classification , Dendritic Cells/chemistry , Dendritic Cells/classification , Dendritic Cells/cytology , Hematopoiesis/physiology , Hematopoietic Stem Cells/chemistry , Hematopoietic Stem Cells/classification , Leukocyte Common Antigens/analysis , Membrane Proteins/analysis , Mice , Mice, Inbred BALB C , Specific Pathogen-Free Organisms
8.
J Immunol ; 166(3): 1531-9, 2001 Feb 01.
Article in English | MEDLINE | ID: mdl-11160193

ABSTRACT

T helper cell-driven activation of murine B cells has been shown to depend upon CD40-CD40 ligand (CD40L) interactions and a defined set of cytokines. These observations are primarily based on the use of conventional B cells obtained from the spleen. Therefore, it is presently unclear whether all mature B cell subsets found in the mouse have an equal dependence upon CD40-CD40L interactions and use the same T cell-derived cytokines. The present study tested the response of splenic follicular and marginal zone as well as peritoneal B2 and B1 B cells to Th cell stimulation. Splenic and peritoneal B cell subsets were sort purified based on CD23 expression, and cultured with rCD40L and cytokines or Th2 cells. The results demonstrate that follicular, marginal zone, and peritoneal B2 B cells require CD40-CD40L interactions and preferentially use IL-4 for optimal proliferation, differentiation, and isotype switching. In contrast, peritoneal B1 B cells use IL-5 in conjunction with CD40-CD40L interactions for maximal Th cell-dependent responses. Furthermore, B1 B cells are capable of proliferating, differentiating, and isotype switching in the absence of CD40-CD40L interactions. B1 B cells are able to respond to Th2 clones in the presence of anti-CD40L mAb as well as to Th2 clones derived from CD40L(-/-) mice. The CD40-CD40L-independent response of B1 B cells is attributable to the presence of both IL-4 and IL-5, and may explain the residual Ab response to T cell-dependent Ags in CD40L- or CD40-deficient mice, and in X-linked hyper-IgM (X-HIM) patients.


Subject(s)
B-Lymphocyte Subsets/immunology , Interleukin-5/physiology , Lymphocyte Activation/immunology , Th2 Cells/immunology , Animals , Antibodies, Blocking/pharmacology , Ascitic Fluid/cytology , Ascitic Fluid/immunology , Ascitic Fluid/metabolism , B-Lymphocyte Subsets/cytology , B-Lymphocyte Subsets/metabolism , CD40 Antigens/biosynthesis , CD40 Ligand/genetics , CD40 Ligand/immunology , CD40 Ligand/pharmacology , Cell Differentiation/genetics , Cell Differentiation/immunology , Cells, Cultured , Clone Cells , Coculture Techniques , Female , Immune Sera/pharmacology , Immunoglobulin Class Switching/immunology , Immunoglobulins/biosynthesis , Interleukin-4/pharmacology , Interleukin-5/immunology , Interleukin-5/pharmacology , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Knockout , Recombinant Proteins/pharmacology , Th2 Cells/metabolism
9.
Cell Immunol ; 203(1): 55-65, 2000 Jul 10.
Article in English | MEDLINE | ID: mdl-10915562

ABSTRACT

Imiquimod and R-848 are members of a family of immune response modifiers that stimulate cytokine production in monocyte/macrophages and dendritic cell cultures. This study evaluated the effects of the imidazoquinolines, imiquimod and R-848, on B lymphocyte activation. Both agents induced proliferation of murine T-cell-depleted and highly purified splenic B cell preparations as well as purified human B cells. Resting and activated B cells responded to these agents, with activated cells responding more efficiently. B cells from the LPS-hyporesponsive C3H/HeJ mice and guanosine-hyporesponsive SJL mice proliferated in response to imiquimod and R-848, indicating a different mechanism of action than lipopolysaccharide and guanine nucleosides. B cells were also stimulated by imiquimod and R-848 to produce increased immunoglobulin levels. Increased expression of a number of B cell activation markers were seen following imiquimod or R-848 stimulation. Finally, R-848 was shown to act as a vaccine adjuvant enhancing OVA-specific IgG2a levels while suppressing total IgE. These results indicate that R-848 and imiquimod are potent activators of B lymphocytes and are capable of augmenting antigen-specific immunoglobulin production.


Subject(s)
Adjuvants, Immunologic/pharmacology , Aminoquinolines/pharmacology , B-Lymphocytes/immunology , Imidazoles/pharmacology , Lymphocyte Activation/drug effects , Animals , Antibody Specificity , Antigens, CD/immunology , B-Lymphocytes/drug effects , B7-2 Antigen , Cells, Cultured , Guanosine/immunology , Humans , Imiquimod , Immunoglobulin Class Switching , Immunoglobulin E/biosynthesis , Immunoglobulin E/immunology , Immunoglobulin G/biosynthesis , Immunoglobulin G/immunology , Lipopolysaccharides/immunology , Membrane Glycoproteins/immunology , Mice , Mice, Inbred Strains , Ovalbumin/immunology , Spleen/cytology , Spleen/immunology
10.
J Immunol ; 164(11): 5729-38, 2000 Jun 01.
Article in English | MEDLINE | ID: mdl-10820250

ABSTRACT

Germinal centers (GCs) are inducible lymphoid microenvironments that support the generation of memory B cells, affinity maturation, and isotype switching. Previously, phenotypic transitions following in vivo B cell activation have been exploited to discriminate GC from non-GC B cells in the mouse and to delineate as many as seven distinct human peripheral B cell subsets. To better understand the differentiative processes occurring within murine GCs, we sought to identify subpopulations of GC B cells corresponding to discrete stages of GC B cell ontogeny. We performed multiparameter flow-cytometric analyses of GC B cells at consecutive time points following immunization of BALB/c mice with SRBC. We resolved the murine GC compartment into subsets based on the differential expression of activation markers, surface Ig isotypes, and differentiation Ags. Class-switched and nonswitched GC B cells emerged contemporaneously, and their relative frequencies remained nearly constant throughout the GC reaction, perhaps reflecting the establishment of a steady state. A significant percentage of the nonswitched B cells with a GC phenotype exhibited surface markers associated with naive B cells, including CD23, surface IgD, and high levels of CD38 consistent with either prolonged recruitment into the GC reaction or protracted expression of these markers during differentiation within the GC. Expression of the activation marker BLA-1 was dynamic over time, with all GC B cells being positive early after immunization, followed by progressive loss as the GC reaction matured into the second and third week. Implications of these results concerning GC evolution are discussed.


Subject(s)
Antigens, CD , Antigens, Differentiation, B-Lymphocyte/biosynthesis , B-Lymphocyte Subsets/immunology , B-Lymphocyte Subsets/metabolism , Germinal Center/cytology , Immunoglobulin Isotypes/biosynthesis , Receptors, Antigen, B-Cell/biosynthesis , ADP-ribosyl Cyclase , ADP-ribosyl Cyclase 1 , Animals , Antigens, Differentiation/biosynthesis , Female , Germinal Center/immunology , Germinal Center/metabolism , Immunization , Immunoglobulin D/biosynthesis , Immunoglobulin G/biosynthesis , Immunoglobulin M/biosynthesis , Kinetics , Leukocyte Common Antigens/biosynthesis , Lymphocyte Activation , Membrane Glycoproteins , Mice , Mice, Inbred BALB C , Mice, Knockout , Microscopy, Confocal , NAD+ Nucleosidase/biosynthesis , Peanut Agglutinin/biosynthesis , Receptors, IgE/biosynthesis
11.
J Allergy Clin Immunol ; 104(6): 1258-64, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10589010

ABSTRACT

BACKGROUND: Oligodeoxynucleotides (ODNs) containing the dinucleotide CpG in a specific sequence context (CpG-ODNs) have the ability to prevent the development of eosinophilic airway inflammation and bronchial hyperreactivity in a murine model of asthma. We have previously demonstrated that CpG-ODNs stimulate expression of the T(H1)-inducing cytokines IFN-gamma and IL-12 in a murine model of asthma and that this stimulation is associated with the protection against asthmatic inflammation. OBJECTIVE: The purpose of this study was to examine whether the protection conferred by CpG-ODNs in a schistosome egg-egg antigen murine model of asthma is dependent on the induction of IFN-gamma, IL-12, or both. METHODS: C57BL/6 mice were sensitized to schistosome eggs in the presence or absence of CpG-ODNs or control ODNs and then stimulated with soluble egg antigen in the airway. The protection offered by CpG-ODNs in these mice was compared with the protection induced by CpG-ODNs in IL-12 and IFN-gamma knockout mice and in mice treated with anticytokine blocking antibodies. Double-knockout mice (IL-12/IFN-gamma) were also generated and used in these studies. Determinations included airway eosinophilic inflammation and bronchial hyperreactivity to inhaled methacholine. RESULTS: We found that CpG-ODNs confer protection against both airway eosinophilia and bronchial hyperreactivity in the absence of IFN-gamma or IL-12 or in the presence of both cytokines together. However, in the absence of either IL-12 or IFN-gamma, mice require 10 times as much CpG-ODNs to be protected against the induction of airway eosinophilia. The T(H2) cytokines IL-4 and IL-5 were reduced in all of the CpG-treated mice, although less in the absence of IL-12 and IFN-gamma. CONCLUSION: These data indicate that CpG-ODNs prevent the generation of T(H2)-like immune responses by multiple mechanisms, which involve, but do not require, IL-12 and IFN-gamma. A direct suppressive effect of CpG-ODNs on T(H2) responses is suggested by their reduction in IFN-gamma and IL-12 knockout mice.


Subject(s)
CpG Islands/physiology , Cytokines/physiology , Animals , Asthma/physiopathology , Asthma/prevention & control , Bronchial Hyperreactivity/prevention & control , Disease Models, Animal , Eosinophilia/prevention & control , Female , Inflammation/prevention & control , Interferon-gamma/physiology , Interleukin-12/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Respiratory Tract Diseases/prevention & control , Th1 Cells/chemistry , Th2 Cells/chemistry
12.
J Immunol ; 161(8): 3880-9, 1998 Oct 15.
Article in English | MEDLINE | ID: mdl-9780153

ABSTRACT

The leukocyte-specific adapter protein SLP-76 is known to augment the transcriptional activity of nuclear factor of activated T cells and AP-1 following TCR ligation. A role for SLP-76 in additional receptor-mediated signaling events is less clear. To define the pattern of SLP-76 expression during murine hemopoiesis, we stained cells isolated from various tissues with a combination of surface markers followed by intracellular staining with a fluorochrome-labeled SLP-76-specific Ab. In the bone marrow, SLP-76 expression is largely restricted to cells of granulocyte and monocyte lineage. Heterogeneous SLP-76 expression is first detected in the CD44+ CD25- subset within the CD3- CD4- CD8- thymocyte population. Interestingly, SLP-76 expression increases as thymocyte maturation progresses within the CD4- CD8- compartment but decreases as cells mature to a CD4+ CD8+ phenotype. SLP-76 expression is then up-regulated following selection and concomitant with maturation to a CD4+ or CD8+ phenotype. In the periphery, SLP-76 is expressed in T lymphocytes with no detectable expression in the B cell compartment. Exposure to the superantigen staphylococcal enterotoxin B augments SLP-76 expression in the reactive T cell subset. Furthermore, in vitro stimulation with TCR-specific Abs augments the existing levels of SLP-76. These data reveal that SLP-76 expression is coordinately regulated with surface expression of a pre-TCR or mature TCR complex during thymocyte development and that TCR ligation elicits signals that result in increased expression of SLP-76.


Subject(s)
Granulocytes/immunology , Hematopoietic Stem Cells/immunology , Lymphatic System/immunology , Monocytes/immunology , Phosphoproteins/immunology , T-Lymphocytes/immunology , Adaptor Proteins, Signal Transducing , Animals , Cell Differentiation/immunology , Cell Lineage/immunology , Female , Granulocytes/cytology , Hematopoietic Stem Cells/cytology , Lymphatic System/cytology , Mice , Mice, Inbred BALB C , Monocytes/cytology , Phosphoproteins/biosynthesis , T-Lymphocytes/cytology
13.
J Immunol ; 160(2): 870-6, 1998 Jan 15.
Article in English | MEDLINE | ID: mdl-9551923

ABSTRACT

Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligodeoxynucleotides (ODN) cause B cell proliferation and Ig secretion, monocyte cytokine secretion, and activation of NK cell lytic activity and IFN-gamma secretion in vivo and in vitro. The potent immune activation by CpG ODN suggests possible utility for enhancing immune responses to vaccines. Mice immunized with recombinant hepatitis B virus surface Ag and a CpG ODN as an immune enhancer have titers of Abs against HBsAg (anti-HBs) that are five times higher than those of mice immunized with HBsAg and the standard adjuvant, aluminum hydroxide (alum). Ab titers in mice immunized with HBsAg and both CpG ODN plus alum were 35 times higher than the titers in mice immunized with alum alone, indicating a strong synergistic interaction between the CpG ODN and alum. ODN without CpG motifs had little or no immune-enhancing activity at the doses used herein. Alum induces a Th2 humoral response (mostly IgG1) and no CTL. In contrast, CpG ODN gives a strong Thl response with predominantly IgG2a Abs and CTL, even when mixed with alum. In vitro studies to determine possible mechanisms of CpG immune-enhancing effects show that CpG ODN induce expression of costimulatory molecules on Ag-presenting cells and drive B cell isotype switching in the appropriate cytokine milieu. These studies demonstrate that CpG ODN are promising new immune enhancers for vaccination applications.


Subject(s)
Adjuvants, Immunologic/genetics , CpG Islands/immunology , Hepatitis B Surface Antigens/genetics , Hepatitis B Surface Antigens/immunology , Vaccines, DNA/immunology , Adjuvants, Immunologic/administration & dosage , Alum Compounds/pharmacology , Animals , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Cells, Cultured , Cytotoxicity, Immunologic/drug effects , Female , Hepatitis B Surface Antigens/administration & dosage , Immunoglobulin Class Switching/drug effects , Immunoglobulin G/biosynthesis , Lymphocyte Activation/drug effects , Mice , Mice, Inbred BALB C , Oligodeoxyribonucleotides/administration & dosage , Oligodeoxyribonucleotides/genetics , Oligodeoxyribonucleotides/immunology , Recombinant Proteins/administration & dosage , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Th1 Cells/immunology , Th1 Cells/metabolism , Vaccines, DNA/administration & dosage , Vaccines, DNA/genetics
14.
J Immunol ; 160(6): 2555-9, 1998 Mar 15.
Article in English | MEDLINE | ID: mdl-9510150

ABSTRACT

Asthma has been increasing in industrialized countries. Evidence suggests that asthma is caused by a Th2 immune response to inhaled environmental Ags and that childhood infections protect against this. We have shown that bacterial DNA contains motifs, centered on unmethylated CpG dinucleotides, which induce Th1-type responses. We hypothesized that the Th1 effect of these CpG motifs may oppose the Th2 type allergic response and suggest that this may account for the protective effect of childhood infection against asthma. We examined the effects of CpG-motif oligodeoxynucleotides (CpG ODN) in a murine model of asthma. Airway eosinophilia, Th2 cytokine induction, IgE production, and bronchial hyperreactivity were prevented by coadministration of CpG ODN with the Ag. Significantly, in a previously sensitized mouse, CpG ODN can prevent allergen-induced airway inflammation. These studies suggest that exposure to CpG DNA may protect against asthma.


Subject(s)
Asthma/drug therapy , Oligodeoxyribonucleotides/pharmacology , Animals , Bronchial Hyperreactivity/drug therapy , Cytokines/biosynthesis , Dinucleoside Phosphates , Immunoglobulin E/blood , Mice , Mice, Inbred C57BL , Th1 Cells/physiology , Th2 Cells/physiology
15.
J Immunol ; 159(3): 1557-69, 1997 Aug 01.
Article in English | MEDLINE | ID: mdl-9233655

ABSTRACT

Human complement receptors type 1 (hCR1;CD35) and type 2 (hCR2;CD21) are expressed on B lymphocytes at specific stages during differentiation and activation. These receptors play critical roles in the immune response to T-dependent Ags in addition to germinal center formation. Expression of both hCR2 and hCR1 is decreased on B lymphocytes of patients with systemic lupus erythematosus (SLE). We have studied the expression of mouse CR2 and CR1 on normal populations of mouse B lymphocytes in BALB/c mice. Our results demonstrate that expression of these receptors in the normal state closely parallels that of hCR2. During bone marrow development, expression is first detected on low B220/high IgM cells, demonstrating that complement receptors appear after central tolerance mechanisms are completed. In the splenic microenvironment the highest levels of receptor expression are found on marginal zone B lymphocytes. Mouse CR2 and CR1 are also found on peritoneal B1a and B1b cells in addition to IgA+ Peyer's patch B cells. Activation of splenic B cells under Th2 conditions results in a marked decrease in receptor expression. To determine whether the patterns of receptor expression also parallel those found in human disease, we studied the MRL lpr/lpr (MRL/lpr) model of SLE. Interestingly, we found an early decrease in complement receptor expression that is progressive and first detectable before major clinical manifestations of nephritis. We hypothesize that the early decrease in complement receptor expression such as that demonstrated by MRL/lpr mice plays an important role in the pathogenesis of murine and perhaps human SLE.


Subject(s)
Autoimmune Diseases/etiology , B-Lymphocyte Subsets/metabolism , Receptors, Complement 3b/biosynthesis , Receptors, Complement 3d/biosynthesis , Animals , Autoimmune Diseases/metabolism , B-Lymphocyte Subsets/immunology , Blotting, Western , Bone Marrow/immunology , Bone Marrow/metabolism , Bone Marrow Cells , Immunoglobulin Switch Region , Lymphocyte Activation/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred MRL lpr , Peritoneal Cavity/cytology , Species Specificity , Spleen/cytology , Spleen/immunology , Spleen/metabolism , T-Lymphocyte Subsets/metabolism , Thymus Gland/cytology
16.
Am J Reprod Immunol ; 37(5): 384-90, 1997 May.
Article in English | MEDLINE | ID: mdl-9196797

ABSTRACT

PROBLEM: Transient involution of the maternal thymus in mice is known to occur during pregnancy. We have previously reported that the hormone responsible for this involution is estrogen. Interestingly, although estrogen crosses the placenta, fetal thymus gland enlarges with advancing gestational age. It is not known if fetal thymocytes are resistant to estrogen or if there are other factors that prevent estrogen from exerting an effect on the development of fetal thymocytes. Therefore we studied the effect of estrogen on isolated fetal thymic glands in vitro. METHOD OR STUDY: Pregnant Balb/c mice were sacrificed at 15 days gestation and fetal thymic lobes were obtained from all fetuses. The glands were cultured in vitro using either control medium or medium to which estrogen was added in two concentrations of 0.5 mg/ 100 ml and 1.0 mg/100 ml. After 12 days of organ culture, total thymocyte counts and phenotypic analysis by three color flow cytometry were performed by using monoclonal antibodies to surface markers of T cells subsets. RESULTS: Estrogen treatment caused a marked suppression of the total number of fetal thymocytes. All CD4 and CD8 defined T cell subsets were reduced with a disproportionate loss of CD4+ single positive (SP), CD8+ SP: CD4+CD8+ double positive (DP) cells. The early thymocyte developmental stages, based on CD44 and CD25 expression, revealed the CD4-CD8-CD3- triple negative compartment (TN) to be composed of almost entirely the earliest population (CD44+CD25-) with the remaining maturational stages depleted. CONCLUSIONS: This study demonstrates that fetal thymus removed from the intact fetus is susceptible to the inhibitory effects of estrogen. Since the fetal thymus enlarges with advanced gestational age, it is clear that the intact fetus invokes a regulatory mechanism which neutralizes the anti-lymphopoietic action of estrogen observed in the adult female.


Subject(s)
Embryonic and Fetal Development/immunology , Estrogens/pharmacology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Thymus Gland/drug effects , Thymus Gland/immunology , Animals , Antigens, CD/immunology , Female , Fetus/drug effects , Fetus/immunology , Flow Cytometry , Mice , Mice, Inbred BALB C , Organ Culture Techniques , Pregnancy , T-Lymphocyte Subsets/immunology
17.
J Immunol ; 158(9): 4211-22, 1997 May 01.
Article in English | MEDLINE | ID: mdl-9126982

ABSTRACT

The expression patterns of the Ly-6C Ag were examined on splenic and thymic lymphocyte subsets of Ly-6.1 and Ly-6.2 strains of mice using the rat mAb 15.1. Ly-6C is expressed on subsets of CD4+ and CD8+ splenocytes, and a portion of NK cells. Within the splenic and lymph node CD4+ T cell compartment, Ly-6C expression is restricted to Ly-6.2 strains of mice, and is present on a subset of naive cells. Ly-6C is expressed on the majority of peripheral CD8+ T cells in both Ly-6.1 and Ly-6.2 strains, and is found primarily on the Ag-experienced subset. In the thymus, Ly-6C is present on subpopulations of CD4- CD8+, CD4- CD8-, and CD4+ CD8- cells. Ly-6C+ CD4- CD8+ thymocytes show a mature phenotype, while Ly-6C+ CD4- CD8- and Ly-6C+ CD4+ CD8- thymocytes appear to be part of the recently described NK1.1+ alphabeta TCR+ population. On account of the marked differences in Ly-6C expression on peripheral CD4+ T cells from Ly-6.1 and Ly-6.2 strains of mice, additional experiments were undertaken to assess Ly-6C expression in parental and Ly-6.1 x Ly-6.2 F1 mice. Neither phenotype dominated in the F1 offspring, with frequencies of Ly-6C+ CD4+ splenocytes falling in the intermediate range. Further experiments compared the staining patterns of the rat anti-pan Ly-6C (Ly-6.1 and Ly-6.2) Ab with a mouse anti-Ly-6.2 allotype specific Ab, with emphasis on both Ly-6.2 and Ly-6.1 x Ly-6.2 F1 mice. The results demonstrate the presence of lymphocytes that express the pan form of Ly-6C but not the form recognized by the alloantibody. This latter finding suggests the presence of more than one form of the Ly-6C Ag.


Subject(s)
Antigens, Ly/metabolism , Membrane Glycoproteins , T-Lymphocyte Subsets/metabolism , Alleles , Animals , Antibodies, Monoclonal , Antigens, CD/analysis , CD24 Antigen , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Epitopes , Flow Cytometry , Histocompatibility Antigens Class I/analysis , Hyaluronan Receptors/analysis , Immunophenotyping , Killer Cells, Natural/immunology , Mice , Mice, Inbred Strains , Rats , Receptors, Antigen, T-Cell, alpha-beta/analysis , Receptors, Antigen, T-Cell, gamma-delta/analysis , Receptors, Interleukin-2/analysis , Spleen/cytology , Thymus Gland/cytology
18.
Cancer Res ; 57(9): 1758-68, 1997 May 01.
Article in English | MEDLINE | ID: mdl-9135020

ABSTRACT

Of six prostatic carcinoma cell lines examined (ALVA31, DU145, JCA1, LNCaP, ND1, and PC3) by flow cytometric analysis, all were found to be positive for Fas antigen. Furthermore, of the prostate tissue specimens studied (six cases), all revealed Fas expression in benign and malignant epithelial cells. The agonistic anti-Fas monoclonal antibody (IPO-4) induced apoptosis in only two of six cell lines investigated, PC3 and ALVA31. PCR analysis indicated that all cell lines expressed normal transmembrane and death domains of Fas antigen. Using Western blot analysis, we found abundant expression of p53 in the cytoplasm of two Fas-resistant cell lines, DU145 and ND1, and did not find p53 in two Fas-sensitive cell lines, PC3 and ALVA31. Western blot and PCR analysis did not show consistent differences between cell lines examined in the expression of Bcl-2, Bcl-X(L), Bcl-X(S), and Bak. In contrast, Bax protein was not detected in two Fas-resistant cell lines, DU145 and ND1. We also showed that three Fas-resistant cell lines, DU145, ND1, and JCA1, expressed CD40, whereas the two Fas-sensitive cell lines, PC3 and ALVA31, were CD40 negative. Fas-sensitive cell lines were transfected with the cDNA encoding CD40, and the CD40-positive transfectant became more resistant to growth inhibition mediated by treatment with TNF-alpha and anti-Fas monoclonal antibody. Treatment with cycloheximide converted the phenotype of resistant cell lines from Fas resistant to Fas sensitive. Moreover, anti-Fas treatment of both resistant and sensitive cell lines induced rapid tyrosine phosphorylation or dephosphorylation of multiple proteins. These results suggest that the apoptotic machinery involved in DNA fragmentation is already in place in Fas-resistant cell lines, and thus, Fas-mediated apoptosis could be a target for therapeutic intervention.


Subject(s)
Apoptosis , Prostatic Neoplasms/pathology , fas Receptor/physiology , Antigen-Antibody Reactions , CD40 Antigens/genetics , CD40 Antigens/metabolism , Cell Division , Cycloheximide/pharmacology , DNA Fragmentation , Flow Cytometry , Humans , Male , Phosphotyrosine/metabolism , Prostatic Neoplasms/metabolism , Protein Tyrosine Phosphatases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Signal Transduction , Transfection , Tumor Cells, Cultured , Tumor Suppressor Protein p53/metabolism , bcl-2-Associated X Protein , bcl-X Protein
19.
Am J Reprod Immunol ; 36(5): 269-77, 1996 Nov.
Article in English | MEDLINE | ID: mdl-8955504

ABSTRACT

PROBLEM: Pregnancy and estrogen are known to suppress B lymphopoiesis as well as lead to thymic involution in the mouse. Additionally, estrogen deficiency by oophorectomy reportedly causes a selective increase in the B220+ B cells in the murine bone marrow. The purpose of this study was to determine if estrogens played a regulatory role in T cell development. METHODS: The first experimental group consisted of 5-6-week-old Balb/c mice that received subcutaneous pellets of placebo, estriol, estradiol, or progesterone. The thymus glands were examined 2-4 weeks after treatment. The second group consisted of 6-week-old Balb/c mice who underwent either bilateral oophorectomy or a sham procedure. Two weeks after the surgery, extensive phenotypic characterization of the thymus and spleen cells was performed by flow cytometry using monoclonal antibodies to surface markers of T cell subsets. RESULTS: Estrogen treatment causes a dramatic reduction of thymic size and cellularity. All defined T cell subsets of CD4 and CD8 were reduced, with a disproportionate loss of CD4+CD8+ double positive cells. Examination of the triple negative (CD3-CD4-CD8-) subset revealed a striking loss of TN developmental progression of the early precursor cells. Based on the expression of CD44 (pgp-1) and CD25 (IL-2R alpha) markers, the TN thymic compartment was composed almost entirely of the earliest population (CD44+, CD25-), with the remaining maturational stages (CD44+, CD25+; CD44-, CD25+; CD44-, CD25-) depleted. In contrast, all T cell developmental stages in the thymus were found to be in normal proportions in the oophorectomized mice, with no differences in the splenic T and B cell subsets. CONCLUSIONS: The study demonstrates that estrogen but not progesterone blocks T cell development in the thymus. However, contrary to our expectation, estrogen deprivation by oophorectomy does not enhance T cell development.


Subject(s)
Estrogens/physiology , T-Lymphocytes/cytology , Thymus Gland/cytology , Animals , Cell Division/drug effects , Female , Flow Cytometry , Mice , Mice, Inbred BALB C , Organ Size/drug effects , Ovariectomy , Progesterone/pharmacology , T-Lymphocyte Subsets/cytology , Thymus Gland/anatomy & histology , Time Factors
20.
Int Immunol ; 8(7): 1121-9, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8757957

ABSTRACT

Previous studies have established the distribution, biochemistry and functional attributes of human CD22, a B cell-restricted glycoprotein. Recently, molecular cloning of the murine CD22 equivalent revealed this molecule to be the same as the previously described Lyb8 alloantigen. Using the anti-Lyb8 mAb Cy34.1.2, the present report documents the expression patterns of CD22 within the murine B cell compartment. The results demonstrate that in the bone marrow, murine CD22 is absent on the surface of pro-B cells, pre-B cells and newly emerging IgM+ B cells. CD22 is present at a low density on immature IgMhi B cells and fully expressed on mature recirculating B cells. In the periphery, murine CD22 is expressed at mature levels on all B cell subsets including follicular, marginal zone, B1 and switched B cells. Further studies showed CD22 to be retained on activated murine B cells for extended periods. Finally, in combination with CD23 and heat stable antigen, CD22 can be used to delineate the immature splenic B cells, and distinguish them from follicular and marginal zone cells. Together, the results demonstrate murine CD22 to be a useful pan marker for all mature B cell subsets.


Subject(s)
Antigens, CD/biosynthesis , Antigens, Differentiation, B-Lymphocyte/biosynthesis , B-Lymphocyte Subsets/metabolism , Cell Adhesion Molecules , Lectins , Membrane Glycoproteins , Animals , Bone Marrow/metabolism , Bone Marrow Cells , CD24 Antigen , Flow Cytometry , Gene Rearrangement, B-Lymphocyte , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Peritoneum/cytology , Peritoneum/metabolism , Receptors, IgE/biosynthesis , Sialic Acid Binding Ig-like Lectin 2 , Spleen/cytology , Spleen/metabolism
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