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1.
Leukemia ; 27(1): 170-82, 2013 Jan.
Article in English | MEDLINE | ID: mdl-22713648

ABSTRACT

Chronic lymphocytic leukemia (CLL) can be immunosuppressive in humans and mice, and CLL cells share multiple phenotypic markers with regulatory B cells that are competent to produce interleukin (IL)-10 (B10 cells). To identify functional links between CLL cells and regulatory B10 cells, the phenotypes and abilities of leukemia cells from 93 patients with overt CLL to express IL-10 were assessed. CD5(+) CLL cells purified from 90% of the patients were IL-10-competent and secreted IL-10 following appropriate ex vivo stimulation. Serum IL-10 levels were also significantly elevated in CLL patients. IL-10-competent cell frequencies were higher among CLLs with IgV(H) mutations, and correlated positively with TCL1 expression. In the TCL1-transgenic (TCL1-Tg) mouse model of CLL, IL-10-competent B cells with the cell surface phenotype of B10 cells expanded significantly with age, preceding the development of overt, CLL-like leukemia. Malignant CLL cells in TCL1-Tg mice also shared immunoregulatory functions with mouse and human B10 cells. Serum IL-10 levels varied in TCL1-Tg mice, but in vivo low-dose lipopolysaccharide treatment induced IL-10 expression in CLL cells and high levels of serum IL-10. Thus, malignant IL-10-competent CLL cells exhibit regulatory functions comparable to normal B10 cells that may contribute to the immunosuppression observed in patients and TCL1-Tg mice.


Subject(s)
B-Lymphocytes, Regulatory/immunology , B-Lymphocytes/immunology , Interleukin-10/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins/physiology , Animals , B-Lymphocytes/metabolism , B-Lymphocytes/pathology , B-Lymphocytes, Regulatory/metabolism , B-Lymphocytes, Regulatory/pathology , Cells, Cultured , Fluorescent Antibody Technique , Humans , Immunosuppression Therapy , Interleukin-10/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Lipopolysaccharides/pharmacology , Mice , Mice, Transgenic
2.
Am J Transplant ; 10(12): 2596-603, 2010 Dec.
Article in English | MEDLINE | ID: mdl-21070606

ABSTRACT

In murine models, the adoptive transfer of CD4(+) /CD25(+) regulatory T cells (T(regs) ) inhibited graft-versus-host disease (GvHD). Previous work has indicated a critical role for the adhesion molecule L-selectin (CD62L) in the function of T(regs) in preventing GvHD. Here we examined the capacity of naive wild-type (WT), CD62L(-/-) and ex vivo expanded CD62L(Lo) T(regs) to inhibit acute GvHD. Surprisingly, we found that CD62L(-/-) T(regs) were potent suppressors of GvHD, whereas CD62L(Lo) T(regs) were unable to inhibit disease despite being functionally competent to suppress allo T cell responses in vitro. Concomitant with improved outcomes, WT and CD62L(-/-) T(regs) significantly reduced liver pathology and systemic pro-inflammatory cytokine production, although CD62L(-/-) T(regs) were less effective in reducing lung pathology. While accumulation of CD62L(-/-) T(regs) in GvHD target organs was equivalent to WT T(regs) , CD62L(-/-) T(regs) did not migrate as well as WT T(regs) to peripheral lymph nodes (PLNs) over the first 2 weeks posttransplantation. This work demonstrated that CD62L was dispensable for T(reg) -mediated protection from GvHD.


Subject(s)
Bone Marrow Transplantation/immunology , Graft vs Host Disease/prevention & control , L-Selectin/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Cell Migration Assays , Male , Mice , Mice, Inbred C57BL , Receptors, Chemokine/biosynthesis
4.
Clin Exp Immunol ; 143(2): 216-27, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16412045

ABSTRACT

Malignant melanoma is often accompanied by a host response of inflammatory cell infiltration that is highly regulated by multiple adhesion molecules. To assess the role of adhesion molecules, including L-selectin and intercellular adhesion molecule-1 (ICAM-1), in this process, subcutaneous primary growth and metastasis to the lung of B16 melanoma cells not expressing L-selectin, ICAM-1 or their ligands were examined in mice lacking L-selectin, ICAM-1 or both. Primary subcutaneous growth of B16 melanoma was augmented by loss of L-selectin, ICAM-1 or both, while pulmonary metastasis was enhanced by the loss of L-selectin or combined loss of L-selectin and ICAM-1. In both situations, the combined loss of L-selectin and ICAM-1 exhibited the greatest effect. This enhancement was associated generally with a reduced accumulation of natural killer (NK) cells, CD4+ T cells and CD8+ T cells and also with a diminished release of interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha but not interleukin (IL)-6. Cytotoxicity against melanoma was not defective by the absence of ICAM-1, L-selectin or both, suggesting that the enhancement of tumour growth and metastasis caused by the loss of adhesion molecules results from an impaired migration of effector cells into the tissue rather than from a suppression of the cytotoxic response. The results indicate that L-selectin and ICAM-1 contribute co-operatively to the anti-tumour reaction by regulating lymphocyte infiltration to the tumour.


Subject(s)
Intercellular Adhesion Molecule-1/immunology , L-Selectin/immunology , Melanoma, Experimental/immunology , Animals , Cell Survival/immunology , Cytokines/immunology , Cytotoxicity, Immunologic/immunology , Immunohistochemistry/methods , Killer Cells, Natural/immunology , Leukocytes/immunology , Lung Neoplasms/secondary , Mice , Mice, Inbred Strains , RNA, Messenger/immunology , T-Lymphocytes/immunology , Tumor Cells, Cultured
5.
Mol Immunol ; 43(6): 550-8, 2006 Feb.
Article in English | MEDLINE | ID: mdl-15936081

ABSTRACT

We have prepared single-chain immunoglobulin Fv fragments from the CD20-specific hybridoma HB13d. One scFv clone demonstrated strong binding to a CD20-derived peptide by ELISA and to CD20-positive cells by flow cytometry, a second had reduced binding, and a third clone did not bind the target antigen. Sequence analysis showed that all three constructs contained shared and unique amino acid changes when compared to the nearest germline match. Molecular modelling of the scFv variants revealed that several of the mutations are located in regions predicted to contact antigen, including a mutation in the heavy chain CDR1 of the strongest binding scFv construct. No similar mutation is present in the highly conserved protein sequences of a number of CD20-specific monoclonal antibodies. BIACORE analysis demonstrated that the mutated scFv had approximately three-fold greater antigen-binding activity than another clone. Competition studies showed that the scFv is able to compete with intact CD20 monoclonal antibody for binding to the target antigen. The improved antigen binding of this scFv will permit the construction of novel CD20-specific reagents for the therapy of lymphomas.


Subject(s)
Antigen-Antibody Reactions/genetics , Antigens, CD20/immunology , Complementarity Determining Regions/genetics , Immunoglobulin Fragments/genetics , Mutation , Amino Acid Sequence , Humans , Hybridomas , Immunoglobulin Fragments/immunology , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region , Models, Molecular
6.
Biochem Soc Trans ; 30(4): 807-11, 2002 Aug.
Article in English | MEDLINE | ID: mdl-12196203

ABSTRACT

CD19 is a B-lymphocyte cell surface molecule that functions as a general response regulator or rheostat, which defines intrinsic and B-cell antigen receptor-induced signalling thresholds that are critical for humoral immunity and expansion of the peripheral B-cell pool. In addition, B-cell responses are influenced by signals transduced through a CD19-CD21 cell surface receptor complex, where the binding of complement C3d to CD21 links humoral immune responses with the innate immune system. This review outlines recent biochemical and genetic studies that characterize the signal transduction pathways utilized by this receptor complex to regulate B-cell intracellular calcium responses.


Subject(s)
Antigens, CD19/immunology , B-Lymphocytes/immunology , Calcium/physiology , Receptors, Complement 3d/immunology , src-Family Kinases/metabolism , Calcium Signaling/physiology , Humans
7.
J Biol Chem ; 276(48): 44820-7, 2001 Nov 30.
Article in English | MEDLINE | ID: mdl-11584010

ABSTRACT

B lymphocyte antigen receptor (BCR) signals are regulated by CD19, with BCR-induced intracellular calcium ([Ca(2+)](i)) responses enhanced by CD19 co-ligation. In this study, CD19 engagement using a dimeric anti-CD19 antibody induced [Ca(2+)](i) mobilization and significantly enhanced BCR-induced [Ca(2+)](i) responses without a requirement for CD19/BCR co-ligation. Although simultaneous CD19 and BCR engagement significantly enhanced CD19/Lyn complex formation and [Ca(2+)](i) responses, downstream tyrosine phosphorylation of CD22 and multiple other cellular proteins was inhibited, as was SHP1 recruitment to phosphorylated CD22. CD19 overexpression also enhanced BCR-induced [Ca(2+)](i) responses, but down-regulated tyrosine phosphorylation of CD22 and multiple other cellular proteins following BCR ligation. Because CD19 and Lyn expression are genetically titrated in B cells, CD19 engagement may augment BCR-induced [Ca(2+)](i) responses by sequestering the available pool of functional Lyn away from downstream negative regulatory proteins such as CD22. Consistent with this, simultaneous CD19 engagement did not further enhance the BCR-induced [Ca(2+)](i) responses of Lyn- or CD22-deficient B cells. Thus, CD19 recruitment of Lyn may preferentially activate selective signaling pathways downstream of the CD19/Lyn complex to the exclusion of other downstream regulatory and effector pathways. Other receptors may also utilize a similar strategy to regulate kinase availability and downstream intermolecular signaling.


Subject(s)
Antigens, CD19/biosynthesis , B-Lymphocytes/metabolism , Calcium/metabolism , Cell Adhesion Molecules , Lectins , src-Family Kinases/physiology , Animals , Antibodies, Monoclonal/metabolism , Antigens, CD/metabolism , Antigens, Differentiation, B-Lymphocyte/metabolism , Blotting, Western , Cell Division , Chromones/pharmacology , Dimerization , Enzyme Inhibitors/pharmacology , Gene Expression Regulation , Immunoglobulin A/metabolism , Immunoglobulin M/metabolism , Lymphocyte Activation , Mice , Morpholines/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Precipitin Tests , Protein Binding , Sialic Acid Binding Ig-like Lectin 2 , Signal Transduction , Spleen/cytology , Time Factors , Tyrosine/metabolism , src-Family Kinases/metabolism
8.
J Immunol ; 167(6): 3190-200, 2001 Sep 15.
Article in English | MEDLINE | ID: mdl-11544305

ABSTRACT

B lymphocytes are critically regulated by signals transduced through the CD19-CD21 cell surface receptor complex, where complement C3d binding to CD21 supplies an already characterized ligand. To determine the extent that CD19 function is controlled by complement activation, CD19-deficient mice (that are hyporesponsive to transmembrane signals) and mice overexpressing CD19 (that are hyperresponsive) were crossed with CD21- and C3-deficient mice. Cell surface CD19 and CD21 expression were significantly affected by the loss of CD21 and C3 expression, respectively. Mature B cells from CD21-deficient littermates had approximately 36% higher cell surface CD19 expression, whereas CD21/35 expression was increased by approximately 45% on B cells from C3-deficient mice. Negative regulation of CD19 and CD21 expression by CD21 and C3, respectively, may be functionally significant because small increases in cell surface CD19 overexpression can predispose to autoimmunity. Otherwise, B cell development and function in CD19-deficient and -overexpressing mice were not significantly affected by a simultaneous loss of CD21 expression. Although CD21-deficient mice were found to express a hypomorphic cell surface CD21 protein at low levels that associated with mouse CD19, C3 deficiency did not significantly affect B cell development and function in CD19-deficient or -overexpressing mice. These results, and the severe phenotype exhibited by CD19-deficient mice compared with CD21- or C3-deficient mice, collectively demonstrate that CD19 can regulate B cell signaling thresholds independent of CD21 engagement and complement activation.


Subject(s)
Antigens, CD19/immunology , B-Lymphocytes/immunology , Complement Activation , Signal Transduction/physiology , Animals , Antigens, CD19/genetics , CD3 Complex/genetics , CD3 Complex/immunology , Exons/genetics , Immunoglobulin D/biosynthesis , Immunoglobulin D/genetics , Immunoglobulin M/biosynthesis , Immunoglobulin M/genetics , Immunophenotyping , Mice , Mice, Inbred C57BL , Mice, Knockout , Rats , Receptors, Complement 3d/genetics , Receptors, Complement 3d/immunology , Specific Pathogen-Free Organisms
9.
Immunogenetics ; 53(5): 357-68, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11486273

ABSTRACT

CD20, the high-affinity IgE receptor beta chain (FcepsilonRIbeta), and HTm4 are structurally related cell surface proteins expressed by hematopoietic cells. Recently, 16 novel human and mouse genes were identified that encode new members of this nascent protein family that we have named the membrane-spanning 4A gene family, with at least 12 subgroups (MS4A1-MS4A12). In the current study, we identified three additional human MS4A genes: MS4A4E, MS4A6E, and MS4A10. All family members have at least four potential transmembrane domains and N- and C-terminal cytoplasmic domains encoded by distinct exons, except MS4A6E which contains two transmembrane domains. Otherwise, the 12 currently identified MS4A genes share common structural features and similar intron/exon splice boundaries, and are clustered along an approximately 600-kb region of Chromosome 11q12. In contrast to other MS4A genes, MS4A4E, MS4A6E, and MS4A10 transcripts were rare and not detected among hematopoietic cells and most nonlymphoid tissues. Sequence polymorphisms were identified in the MS4A6E gene and common splice variants were observed for the MS4A4A, MS4A5, MS4A6A, and MS4A7 genes. Thus, the MS4A family currently includes 24 distinct human and mouse genes. Like CD20 and FcepsilonRIbeta, the 10 other human MS4A family members are likely to be components of oligomeric cell surface complexes involved in signal transduction in diverse cell lineages.


Subject(s)
Cell Cycle Proteins , Chromosomes, Human, Pair 11/genetics , Membrane Proteins/genetics , Multigene Family , Amino Acid Sequence , Antigens, CD20/genetics , Base Sequence , Chromosome Mapping , Evolution, Molecular , Exons , Genetic Linkage , Humans , Introns , Molecular Sequence Data , Receptors, IgE/genetics
10.
J Immunol ; 167(5): 2469-78, 2001 Sep 01.
Article in English | MEDLINE | ID: mdl-11509585

ABSTRACT

CD19 and the Src family protein tyrosine kinases (PTKs) are important regulators of intrinsic signaling thresholds in B cells. Regulation is achieved by cross-talk between Src family PTKs and CD19; Lyn is essential for CD19 phosphorylation, while CD19 establishes an Src family PTK activation loop that amplifies kinase activity. However, CD19-deficient (CD19(-/-)) B cells are hyporesponsive to transmembrane signals, while Lyn-deficient (Lyn(-/-)) B cells exhibit a hyper-responsive phenotype resulting in autoimmunity. To identify the outcome of interactions between CD19 and Src family PTKs in vivo, B cell function was examined in mice deficient for CD19 and Lyn (CD19/Lyn(-/-)). Remarkably, CD19 deficiency suppressed the hyper-responsive phenotype of Lyn(-/-) B cells and autoimmunity characterized by serum autoantibodies and immune complex-mediated glomerulonephritis in Lyn(-/-) mice. Consistent with Lyn and CD19 each regulating conventional B cell development, B1 cell development was markedly reduced by Lyn deficiency, with further reductions in the absence of CD19 expression. Tyrosine phosphorylation of Fyn and other cellular proteins induced following B cell Ag receptor ligation was dramatically reduced in CD19/Lyn(-/-) B cells relative to Lyn(-/-) B cells, while Syk phosphorylation was normal. In addition, the enhanced intracellular Ca(2+) responses following B cell Ag receptor ligation that typify Lyn deficiency were delayed by the loss of CD19 expression. BCR-induced proliferation and humoral immune responses were also markedly inhibited by CD19/Lyn deficiency. These findings demonstrate that while the CD19/Lyn amplification loop is a major regulator of signal transduction thresholds in B lymphocytes, CD19 regulation of other Src family PTKs also influences B cell function and the development of autoimmunity.


Subject(s)
Antigens, CD19/metabolism , Autoimmunity , Signal Transduction/immunology , src-Family Kinases/deficiency , src-Family Kinases/immunology , Animals , Antibody Formation , Antigens, CD19/genetics , B-Lymphocytes/cytology , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Calcium Signaling , Cell Differentiation , Immunoglobulin M/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphorylation , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-fyn , Receptors, Antigen, B-Cell/metabolism , Up-Regulation , src-Family Kinases/genetics
11.
Genomics ; 72(2): 119-27, 2001 Mar 01.
Article in English | MEDLINE | ID: mdl-11401424

ABSTRACT

CD20, high-affinity IgE receptor beta chain (FcepsilonRIbeta), and HTm4 are structurally related cell-surface proteins expressed by hematopoietic cells. In the current study, 16 novel human and mouse genes that encode new members of this nascent protein family were identified. All family members had at least four potential membrane-spanning domains, with N- and C-terminal cytoplasmic domains. This family was therefore named the membrane-spanning 4A gene family, with at least 12 subgroups (MS4A1 through MS4A12) currently representing at least 21 distinct human and mouse proteins. Each family member had unique patterns of expression among hematopoietic cells and nonlymphoid tissues. Four of the 6 human MS4A genes identified in this study mapped to chromosome 11q12-q13.1 along with CD20, FcepsilonRIbeta, and HTm4. Thus, like CD20 and FcepsilonRIbeta, the other MS4A family members are likely to be components of oligomeric cell surface complexes that serve diverse signal transduction functions.


Subject(s)
Cell Cycle Proteins , Membrane Proteins/genetics , Multigene Family , Amino Acid Sequence , Animals , Antigens, CD20/genetics , Gene Expression , Humans , Membrane Proteins/chemistry , Molecular Sequence Data , Protein Structure, Tertiary , Receptors, IgE/genetics , Sequence Homology, Amino Acid
12.
J Allergy Clin Immunol ; 107(6): 1019-24, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11398079

ABSTRACT

BACKGROUND: Airway inflammation and airway hyperresponsiveness (AHR) are fundamental features of asthma. Migration of inflammatory cells from the circulation into the lungs is dependent on adhesion molecule interactions. The cell surface adhesion molecule L-selectin has been demonstrated to mediate leukocyte rolling on inflamed and noninflamed pulmonary endothelium. However, its role in the development of airway inflammation and AHR in asthma has not been examined. OBJECTIVE: We sought to characterize the role of L-selectin in the recruitment of inflammatory cells to the airway-lung and the development of AHR in a murine model of asthma. METHODS: An ovalbumin (OVA)-induced allergic airway disease model of asthma was applied to L-selectin-deficient (LKO) mice and C57BL/6 wild-type (WT) control mice. The development of airway inflammation was assessed by examining leukocyte influx into bronchoalveolar lavage (BAL) fluid and the lung. Total and differential BAL leukocyte counts were determined, and the immunophenotype of BAL lymphocytes was assessed by means of flow cytometry. The development of AHR was assessed by means of whole-body plethysmography. RESULTS: Airway-lung inflammation was equivalent in LKO and WT mice sensitized-challenged with OVA, as measured by total and differential BAL cell counts and histologic analysis of lung tissue. Numbers of eosinophils, neutrophils, lymphocytes, and monocytes in BAL fluid were equivalent in LKO and WT mice. However, phenotypic analysis of BAL lymphocytes demonstrated significantly reduced CD3(+) populations and increased B220(+) populations in LKO compared with WT mice (P <.05). Remarkably, despite a fulminant inflammatory response in the airway-lung in LKO mice sensitized-challenged with OVA, AHR was completely abrogated. CONCLUSION: L-selectin plays a crucial role in the development of AHR but not allergic inflammation in an animal model of asthma. L-selectin represents a potential target for novel asthma therapies specifically aimed at controlling AHR.


Subject(s)
Asthma/physiopathology , Bronchial Hyperreactivity/physiopathology , Disease Models, Animal , Inflammation/physiopathology , L-Selectin/metabolism , Animals , Bronchial Hyperreactivity/immunology , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , Flow Cytometry , Humans , Immunoglobulin E/blood , Immunoglobulin E/immunology , Inflammation/immunology , Leukocyte Count , Lung/pathology , Methacholine Chloride/pharmacology , Mice , Mice, Inbred C57BL , Ovalbumin/immunology , Ovalbumin/pharmacology
13.
J Allergy Clin Immunol ; 107(4): 734-8, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11295667

ABSTRACT

T lymphocytes play a critical role in the development of allergic inflammation in asthma. Early in the allergic response, T lymphocytes migrate from the circulation into the lung to initiate and propagate airway inflammation. The adhesion molecules that mediate lymphocyte entry into inflamed lung have not been defined. This study directly examined the roles of L-selectin and intercellular adhesion molecule 1 (ICAM-1) in lymphocyte migration to the lung during an allergic inflammatory response in an animal model of asthma. Short-term (1 hour) in vivo migration assays and various combinations of adhesion molecule-deficient and wild-type mice were used. Migration of in vivo activated lymphocytes into inflamed lung was significantly greater than entry of resting lymphocytes into noninflamed lung (24.5% +/- 2.7% vs 9.5% +/- 1.3%, P =.001). Migration of activated lymphocytes into inflamed lung was inhibited by 30% in the absence of L-selectin (17.3% +/- 1.3%, P =.04), 47% in the absence of cell surface ICAM-1 (13.0% +/- 2.5%, P =.01), and 47% in the absence of endothelial ICAM-1 (13.0% +/- 2.5%, P =.01). Loss of ICAM-1 on both lymphocytes and lung endothelium inhibited lymphocyte migration by 60% (9.8% +/- 1.8%, P =.002). These findings demonstrate clear roles for both L-selectin and ICAM-1 in lymphocyte migration to the lung during an allergic inflammatory response, with ICAM-1 playing a greater role.


Subject(s)
Asthma/immunology , Disease Models, Animal , Hypersensitivity/immunology , Inflammation/immunology , Intercellular Adhesion Molecule-1/physiology , L-Selectin/physiology , Lung/immunology , T-Lymphocytes/physiology , Animals , Cell Movement , Mice , Mice, Inbred C57BL , Ovalbumin/immunology
14.
J Leukoc Biol ; 69(4): 565-74, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11310842

ABSTRACT

Sulfation is an essential component of the selectin ligands, potentially mediated by members of a new family of carbohydrate sulfotransferases. In this study, we assessed the contributions of CHST1, CHST2, CHST3, and CHST4 in producing functional L-selectin ligands. Human umbilical vein endothelial cells predominantly expressed CHST1 and CHST2 transcripts with low levels of CHST3 mRNA, while cytokine activation up-regulated CHST2 expression and induced low-level CHST4 expression. A human umbilical vein endothelial cell line, EA.hy926, displayed functional L-selectin ligands that correlated with CHST1 and CHST2 expression in the absence of CHST4 expression. Increased CHST1 or CHST2 expression by a cell line expressing low-level L-selectin ligand activity during in vitro flow chamber assays increased rolling leukocyte numbers, reduced rolling velocities, and enhanced leukocyte rolling under higher shear stresses. These results suggest that CHST1 and CHST2 contribute to the generation of optimal L-selectin ligands in vascular endothelial cells at sites of inflammation.


Subject(s)
Chemotaxis, Leukocyte/physiology , Endothelium, Vascular/enzymology , L-Selectin/physiology , Sulfotransferases/biosynthesis , Animals , COS Cells , Cell Adhesion/physiology , Cells, Cultured , Chlorocebus aethiops , DNA, Complementary/genetics , Endothelium, Vascular/cytology , Enzyme Induction , Fucosyltransferases/biosynthesis , Fucosyltransferases/genetics , Humans , Lewis X Antigen/analogs & derivatives , Ligands , Membrane Glycoproteins/metabolism , Oligosaccharides/metabolism , RNA, Messenger/biosynthesis , Recombinant Fusion Proteins/physiology , Rheology , Sialyl Lewis X Antigen/analogs & derivatives , Stress, Mechanical , Sulfotransferases/genetics , Sulfotransferases/physiology , Transfection , Tumor Cells, Cultured , Umbilical Veins , Carbohydrate Sulfotransferases
15.
J Immunol ; 166(8): 4899-907, 2001 Apr 15.
Article in English | MEDLINE | ID: mdl-11290767

ABSTRACT

L-Selectin mediates leukocyte rolling on endothelium and immobilized leukocytes. Its regulation has been the subject of much study, and the conformation of the molecule may play an important role in its function. Here we report that a conformational change in L-selectin, induced by an anti-lectin domain mAb (LAM1-116) and recognized by another mAb directed to a conserved epitope on L-selectin (EL-246), predisposed L-selectin to cytoskeletal association. This effect was due to direct binding of the mAb, not to overt signaling events, and was specific to LAM1-116. Nineteen other anti-L-selectin mAbs directed against the lectin, epidermal growth factor, or short consensus repeat domains lacked this activity. The induced conformational change occurred at 37 degrees C, at 4 degrees C, in the presence of sodium azide and tyrosine kinase inhibitors herbimycin A and genistein, and with soluble detergent-extracted L-selectin. In the presence of LAM1-116, EL-246 induced cytoskeletal association of L-selectin in the absence of Ab cross-linking as visualized by L-selectin staining after low dose detergent treatment of the cells. We propose that the conformational change described herein regulates L-selectin-mediated events by exposing a high avidity binding site that, when engaged, triggers association of L-selectin with the cytoskeleton, which may lead to stronger tethers with physiological ligands.


Subject(s)
Antibodies, Monoclonal/metabolism , Binding Sites, Antibody , Cytoskeleton/immunology , Detergents/pharmacology , Epitopes/metabolism , L-Selectin/immunology , L-Selectin/metabolism , Animals , Antibodies, Monoclonal/analysis , Antibodies, Monoclonal/pharmacology , Benzoquinones , Binding Sites, Antibody/drug effects , Cattle , Cell Fractionation , Cell Line , Cyanogen Bromide , Cytoplasm/genetics , Cytoplasm/immunology , Cytoskeleton/drug effects , Cytoskeleton/genetics , E-Selectin/genetics , E-Selectin/metabolism , Enzyme Inhibitors/pharmacology , Epitopes/biosynthesis , Epitopes/immunology , Genistein/pharmacology , Humans , Immunoglobulin Fab Fragments/pharmacology , L-Selectin/analysis , L-Selectin/genetics , Lactams, Macrocyclic , Leukocytes/chemistry , Leukocytes/drug effects , Leukocytes/enzymology , Leukocytes/immunology , Mice , Microspheres , Octoxynol , P-Selectin/genetics , P-Selectin/metabolism , Polyethylene Glycols/pharmacology , Precipitin Tests , Protein Conformation/drug effects , Protein Structure, Tertiary/genetics , Protein-Tyrosine Kinases/antagonists & inhibitors , Quinones/pharmacology , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Rifabutin/analogs & derivatives , Sheep , Solubility , Staining and Labeling , Transfection , Up-Regulation/drug effects , Up-Regulation/immunology
16.
J Immunol ; 166(4): 2412-9, 2001 Feb 15.
Article in English | MEDLINE | ID: mdl-11160300

ABSTRACT

Systemic lupus erythematosus is an autoimmune disease characterized by the presence of autoantibodies. One of the unique targets of the immune system in systemic lupus erythematosus is Sm, a ribonucleoprotein present in all cells. To understand the regulation of B cells specific to the Sm Ag in normal mice, we have generated an Ig H chain transgenic mouse (2-12H Tg). 2-12H Tg mice produce B cells specific for the Sm that remain tolerant due to ignorance. We demonstrate here that anti-Sm B cells of 2-12H Tg mice can differentiate into Sm-specific peritoneal B-1 cells that remain tolerant. Differentiation to B-1 and tolerance are governed by the strength of B cell receptor signaling, since manipulations of the B cell receptor coreceptors CD19 and CD22 affect anti-Sm B cell differentiation and autoantibody production. These results suggest a differentiation scheme in which peripheral ignorance to Sm is maintained in mice by the differentiation of anti-Sm B cells to B-1 cells that have increased activation thresholds.


Subject(s)
Autoantigens/immunology , B-Lymphocyte Subsets/immunology , B-Lymphocyte Subsets/metabolism , Cell Adhesion Molecules , Immune Tolerance , Lectins , Lupus Erythematosus, Systemic/immunology , Receptors, Antigen, B-Cell/physiology , Ribonucleoproteins, Small Nuclear/immunology , Signal Transduction/immunology , Animals , Antigens, CD/biosynthesis , Antigens, CD/genetics , Antigens, CD19/biosynthesis , Antigens, CD19/genetics , Antigens, Differentiation, B-Lymphocyte/biosynthesis , Antigens, Differentiation, B-Lymphocyte/genetics , Autoantibodies/blood , Autoantibodies/physiology , B-Lymphocyte Subsets/pathology , Cell Differentiation/genetics , Cell Differentiation/immunology , Immune Tolerance/genetics , Mice , Mice, Inbred MRL lpr , Mice, Knockout , Mice, Transgenic , Peritoneum/cytology , Peritoneum/immunology , Receptors, Antigen, B-Cell/biosynthesis , Sialic Acid Binding Ig-like Lectin 2 , Signal Transduction/genetics , Spleen/cytology , Spleen/immunology , snRNP Core Proteins
17.
Int Rev Immunol ; 20(6): 739-62, 2001.
Article in English | MEDLINE | ID: mdl-11913948

ABSTRACT

B lymphocyte development and function depend upon the activity of intrinsic and B cell antigen receptor (BCR)-induced signals. These signals are interpreted, amplified, fine-tuned, or suppressed through the precise actions of specialized cell surface coreceptors, or "response regulators," that inform B cells of their extracellular environment. Important cell surface response regulators include the CD19/CD21 complex, CD22, and CD72. CD19 establishes a novel Src-family protein tyrosine kinase (PTK) amplification loop that regulates basal signaling thresholds and intensifies Src-family PTK activation following BCR ligation. In turn, CD22 limits the intensity of CD19-dependent, BCR-generated signals through the recruitment of potent phosphotyrosine and phosphoinositide phosphatases. Herein we discuss our current understanding of how CD19/CD21 and CD22 govern the emergence and intensity of BCR-mediated signals, and how alterations in these tightly controlled regulatory activities contribute to autoimmunity in mice and humans.


Subject(s)
Antigens, CD19/metabolism , Antigens, CD/metabolism , Antigens, Differentiation, B-Lymphocyte/metabolism , B-Lymphocytes/immunology , Cell Adhesion Molecules , Lectins , Receptors, Complement 3d/metabolism , Animals , Antigens, CD/chemistry , Antigens, CD/genetics , Antigens, CD19/chemistry , Antigens, CD19/genetics , Antigens, Differentiation, B-Lymphocyte/chemistry , Antigens, Differentiation, B-Lymphocyte/genetics , Autoimmunity , Humans , Lymphocyte Activation , Mice , Mice, Knockout , Models, Immunological , Receptors, Antigen, B-Cell/metabolism , Receptors, Complement 3d/chemistry , Receptors, Complement 3d/genetics , Sialic Acid Binding Ig-like Lectin 2 , Signal Transduction , src-Family Kinases/metabolism
18.
J Immunol ; 165(12): 6915-21, 2000 Dec 15.
Article in English | MEDLINE | ID: mdl-11120817

ABSTRACT

Although it is now appreciated that mast cell-mediated release of TNF-alpha is critical for resolution of acute septic peritonitis, questions remain as to how mast cells are activated upon peritoneal bacterial infection. Clues to how this may occur have been derived from earlier studies by Prodeus et al. in which complement proteins C3 and C4 were shown to be required for survival following cecal ligation and puncture (CLP), a model for acute septic peritonitis. To evaluate the mechanism for mast cell activation in the CLP model, complement receptor CD21/CD35-deficient mice (Cr2(null)) were examined in the present study. Along with CD19-deficient (CD19(null)) mice, these animals exhibit decreased survival following CLP compared with wild-type littermates. Injection of IgM before CLP does not change survival rates for Cr2(null) mice and only partially improves survival of CD19(null) mice, implicating CD21/CD35 and CD19 in mast cell activation. Interestingly, early TNF-alpha release is also impaired in Cr2(null) and CD19(null) animals, suggesting that these molecules directly affect mast cell activation. Cr2(null) and CD19(null) mice demonstrate an impairment in neutrophil recruitment and a corresponding increase in bacterial load. Examination of peritoneal mast cells by flow cytometry and confocal microscopy reveals the expression and colocalization of CD21/CD35 and CD19. Taken together, these findings suggest that the engagement of complement receptors CD21/CD35 along with CD19 on the mast cell surface by C3 fragments may be necessary for the full expression of mast cell activation in the CLP model.


Subject(s)
Antigens, CD19/physiology , Mast Cells/immunology , Mast Cells/metabolism , Peritonitis/immunology , Receptors, Complement 3b/physiology , Receptors, Complement 3d/physiology , Sepsis/immunology , Acute Disease , Animals , Antigens, CD19/biosynthesis , Antigens, CD19/genetics , Antigens, CD19/metabolism , Ascitic Fluid/immunology , Ascitic Fluid/metabolism , Ascitic Fluid/pathology , Cecum/surgery , Cell Membrane/immunology , Cell Membrane/metabolism , Flow Cytometry , Leukocyte Count , Ligation , Male , Mast Cells/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Neutrophils/pathology , Peritoneal Lavage , Peritonitis/genetics , Peritonitis/mortality , Proto-Oncogene Proteins c-kit/biosynthesis , Punctures , Receptors, Complement 3b/biosynthesis , Receptors, Complement 3b/metabolism , Receptors, Complement 3d/biosynthesis , Receptors, Complement 3d/genetics , Receptors, Complement 3d/metabolism , Survival Analysis , Tumor Necrosis Factor-alpha/metabolism
19.
Clin Exp Immunol ; 122(3): 484-92, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11122259

ABSTRACT

L-selectin is a leucocyte adhesion molecule involved in leucocyte interactions with vascular endothelial cells. Following leucocyte activation L-selectin is endoproteolytically released from the cell surface. To assess whether psoriasis vulgaris results in systemic leucocyte activation, we examined expression levels of L-selectin on subsets of peripheral blood leucocytes from patients with psoriasis (n = 25) and normal control subjects. Serum levels of soluble L-selectin were quantified by ELISA in patients with psoriasis (n = 75), pustulosis palmaris et plantaris, and contact dermatitis, as well as normal control subjects. Psoriasis severity was evaluated by psoriasis area and severity index (PASI). L-selectin expression levels on CD4+ T cells, B cells, monocytes, and neutrophils from patients with severe-type psoriasis (PASI > or = 15) was significantly decreased compared with leucocytes from normal control subjects. Furthermore, L-selectin expression on CD4+ T cells showed good inverse correlation with PASI scores. Monocyte L-selectin expression was restored when the skin lesions of psoriasis were remitted. The frequencies of L-selectin+ CD4+ T cells or L-selectin+ CD8+ T cells from patients with psoriasis were almost normal. Serum L-selectin levels in patients with severe-type psoriasis were significantly higher than those in normal control subjects. These results suggest that subsets of leucocytes may be activated in psoriasis, and that L-selectin expression levels on some leucocyte subsets, especially CD4+ T cells, tend to correlate with disease severity of psoriasis.


Subject(s)
L-Selectin/metabolism , Leukocytes/metabolism , Psoriasis/metabolism , Adult , Aged , Aged, 80 and over , Enzyme-Linked Immunosorbent Assay/methods , Female , Humans , L-Selectin/blood , Leukocyte Count , Male , Middle Aged , Psoriasis/blood , Psoriasis/physiopathology
20.
J Immunol ; 165(12): 6872-9, 2000 Dec 15.
Article in English | MEDLINE | ID: mdl-11120811

ABSTRACT

c-Abl is a nonreceptor tyrosine kinase that we have recently linked to growth factor receptor signaling. The c-Abl kinase is ubiquitously expressed and localizes to the cytoplasm, plasma membrane, cytoskeleton, and nucleus. Thus, c-Abl may regulate signaling processes in multiple subcellular compartments. Targeted deletion or mutation of c-Abl in mice results in a variety of phenotypes, including splenic and thymic atrophy and lymphopenia. Additionally, lymphocytes isolated from specific compartments of c-Abl mutant mice have reduced responses to a variety of stimuli and an increased susceptibility to apoptosis following growth factor deprivation. Despite these observations, little is known regarding the signaling mechanisms responsible for these phenotypes. We report here that splenic B cells from c-Abl-deficient mice are hyporesponsive to the proliferative effects of B cell Ag receptor (BCR) stimulation. The c-Abl kinase activity and protein levels are elevated in the cytosol following activation of the BCR in B cell lines. We show that c-Abl associates with and phosphorylates the BCR coreceptor CD19, and that c-Abl and CD19 colocalize in lipid membrane rafts. These data suggest a role for c-Abl in the regulation of B cell proliferation downstream of the BCR, possibly through interactions with CD19.


Subject(s)
Antigens, CD19/metabolism , B-Lymphocytes/enzymology , B-Lymphocytes/immunology , Proto-Oncogene Proteins c-abl/metabolism , Receptors, Antigen, B-Cell/metabolism , Animals , Animals, Outbred Strains , B-Lymphocytes/metabolism , Detergents , Enzyme Activation/genetics , Enzyme Activation/immunology , Humans , Lymphocyte Activation/genetics , Membrane Microdomains/immunology , Membrane Microdomains/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Proto-Oncogene Proteins c-abl/biosynthesis , Proto-Oncogene Proteins c-abl/deficiency , Proto-Oncogene Proteins c-abl/genetics , Receptors, Antigen, B-Cell/physiology , Solubility , Substrate Specificity/genetics , Substrate Specificity/immunology , Tumor Cells, Cultured , Up-Regulation/genetics , Up-Regulation/immunology
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