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1.
Genes Immun ; 4(2): 168-9, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12618866

ABSTRACT

A point mutation in exon A (C to G transversion at position 77) of human PTPRC (CD45) has recently been associated with the development of multiple sclerosis (MS) for at least a subgroup of patients. In the present report, we studied the frequency of the 77C-->G transversion in two other autoimmune diseases namely systemic sclerosis (SSc) and systemic lupus erythematosus (SLE). The mutation was found with significantly enhanced frequency in patients suffering from SSc suggesting that PTPRC could play a role as susceptibility gene not only in MS but also in other autoimmune diseases. Further understanding of the mode of interaction of mutant PTPRC with other susceptibility genes may uncover mechanisms common in various autoimmune disorders.


Subject(s)
Genetic Predisposition to Disease , Leukocyte Common Antigens/genetics , Point Mutation , Scleroderma, Systemic/genetics , Exons , Gene Frequency , Humans , Lupus Erythematosus, Systemic/genetics , Multiple Sclerosis/genetics
2.
Blood ; 98(10): 3035-41, 2001 Nov 15.
Article in English | MEDLINE | ID: mdl-11698288

ABSTRACT

Splenectomy increases the number of B cells in the blood of humans and animals. It is unknown whether this is due to changes in migration, proliferation, or both. The numbers of naïve (IgD(+)IgM(+)), memory (IgD(-)IgM(high)), newly formed (IgM(high)CD90(high)), early recirculating follicular (IgM(low)CD90(high)), recirculating follicular (IgM(low)CD90(-)), and marginal zone (IgM(high)CD90(-)) phenotype B cells were determined in control and splenectomized rats by flow cytometry. All subsets increased significantly in the blood after splenectomy. Because surface molecules are involved in the regulation of migration and proliferation, their expression (lymphocyte function-associated antigen 1 [LFA-1], intercellular adhesion molecule 1 (ICAM-1), L-selectin, alpha4-integrins, CD44, major histocompatability complex class II, interleukin 2 receptor-alpha chain) was determined on B- and T-cell subsets of both groups. B cells, but not T cells, showed a significantly reduced LFA-1 and ICAM-1 expression in blood and lymph nodes, whereas the expression of the other surface molecules analyzed remained unchanged. The down-regulation of these molecules did not influence the adherence of B cells to high endothelial venules in vitro. In vivo, however, ICAM-1(low)-expressing B cells migrated significantly faster through lymph nodes (ICAM-1(low) 41 +/- 5 hours versus ICAM-1(high) 58 +/- 3 hours), whereas proliferation of B cells in bone marrow, lymph node, and blood remained unchanged. Thus, the presence of one organ is necessary for appropriate expression of LFA-1 and ICAM-1 on B cells in other, distant organs. The more rapid transit of ICAM-1(low) B cells through lymph nodes may be responsible for the increased B-cell number in the blood after splenectomy.


Subject(s)
B-Lymphocyte Subsets/metabolism , Intercellular Adhesion Molecule-1/biosynthesis , Lymphocyte Function-Associated Antigen-1/biosynthesis , Lymphocytosis/etiology , Splenectomy/adverse effects , Animals , Antigens, CD/biosynthesis , Antigens, CD/genetics , B-Lymphocyte Subsets/immunology , Blood Cells/immunology , Blood Cells/metabolism , Cell Adhesion , Cell Division , Cell Movement , Down-Regulation , Endothelium, Vascular/cytology , Flow Cytometry , Gene Expression Regulation , Histocompatibility Antigens Class II/biosynthesis , Histocompatibility Antigens Class II/genetics , Hyaluronan Receptors/biosynthesis , Hyaluronan Receptors/genetics , Immunophenotyping , Integrin alpha4 , Intercellular Adhesion Molecule-1/genetics , L-Selectin/biosynthesis , L-Selectin/genetics , Lymph Nodes/cytology , Lymphocyte Activation , Lymphocyte Count , Lymphocyte Function-Associated Antigen-1/genetics , Organ Specificity , Rats , Rats, Inbred Lew , Receptors, Interleukin-2/biosynthesis , Receptors, Interleukin-2/genetics , Time Factors
3.
Eur J Immunol ; 31(7): 2217-26, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11449376

ABSTRACT

Transgenic expression of apoptosis-inducing molecules could be a strategy to protect cells and tissues from destruction by apoptosis-susceptible effector T cells. Some evidence for the potency of this approach has been obtained in mouse and rat transplantation models. However, limited data are available on the capacity of apoptosis-inducing molecules to modulate human alloimmune responses. In this study we analyzed the in vitro consequences of an interaction of human T cells with allogeneic 293 cells and 293 transfectants stably expressing high levels of the apoptosis-inducing CD95 ligand (CD95L). Both, CD95L(-) and CD95L(+) 293 cells were able to activate allogeneic T cells as demonstrated by comparable CD25 expression at day 2 of culture. The analysis of viable T cells at day 7, however, revealed anti-293 cytotoxic activity only in cultures that had been stimulated with CD95L(-) 293 cells. Alloactivated effector T cells lysed CD95L(-) and CD95L(+) 293 targets with similar efficiency when tested in a 4-h 51Cr-release assay. Prolongation of the effector phase to 20 h resulted in a further increase in the destruction of CD95L(-) target cells, whereas lysis of CD95L(+) targets remained low. These data suggest that genetically engineered expression of CD95L on cells or tissues could be an approach to control human T cell reactivity towards allografts. During the induction of an alloimmune response depletion of cytotoxic precursor cells may be obtained by overexpressing CD95L on stimulatory cells; CD95L expression on graft tissue might limit T cell-mediated destruction of the transplant during the effector phase of the response.


Subject(s)
Apoptosis , Isoantigens/immunology , Membrane Glycoproteins/physiology , T-Lymphocytes, Cytotoxic/immunology , Cell Line , Cells, Cultured , Cytotoxicity Tests, Immunologic , Down-Regulation , Fas Ligand Protein , Genetic Engineering , Humans , Immune Tolerance , Killer Cells, Natural/immunology , Lymphocyte Activation , Membrane Glycoproteins/genetics , Transfection
4.
Transplantation ; 71(8): 1124-31, 2001 Apr 27.
Article in English | MEDLINE | ID: mdl-11374414

ABSTRACT

BACKGROUND: In clinical organ transplantation monoclonal antibodies (mAb) to different surface molecules of immunocompetent cells become integral parts of the immunosuppressive therapy. In this study, a mAb against the rat leukocyte common antigen CD45 (RT7) was tested for its immunosuppressive potency after a single perioperative injection. METHODS: Binding and depleting properties of the anti-RT7 mAb were investigated by flow cytometry. In the fully major histocompatibility complex-disparate heart and skin transplantation models (LEW [RT1l]--> LEW.1W [RT1u]), a single dose of anti-RT7 mAb (10 mg/kg) was administered intravenously (day -1). To characterize the long-term acceptance of heart allografts second set skin transplantation (day 100), mixed lymphocyte reaction studies (day 100) and reverse transcriptase-polymerase chain reaction analysis for intragraft cytokine expression (day 200) were performed. RESULTS: The anti-RT7 mAb bound to nearly all hematopoietic lineage cells, but particularly T and NK cells, and profoundly depleted these cells in circulation and lymphoid tissues. Anti-RT7 mAb-treated rats showed long-term acceptance of heart allografts (>200 days; n=12), whereas untreated recipients rejected allografts by day 8 (n=6). In contrast to hearts, primary skin allograft survival was only moderately prolonged. Animals with stable heart allograft acceptance showed normal in vitro lymphocyte proliferation responses to donor and third party antigen. These recipients also acutely rejected second set donor-strain skin grafts without inducing rejection of persisting heart allografts. Reverse transcriptase-polymerase chain reaction analysis of intragraft cytokines showed up-regulation of Fas-ligand and IL-4 mRNA in long-surviving heart allografts. CONCLUSIONS: The findings demonstrate that a single injection of an anti-RT7 mAb in the rat can induce stable long-term acceptance of heart allografts by transient but profound T-cell depletion. Local immunoregulatory mechanisms seem to play a role for maintenance of long-term graft acceptance.


Subject(s)
Antibodies, Monoclonal/pharmacology , Graft Survival/immunology , Heart Transplantation/immunology , Leukocyte Common Antigens/immunology , Skin Transplantation/immunology , Animals , B-Lymphocytes/immunology , Flow Cytometry , Graft Survival/drug effects , Granulocytes/immunology , Immunosuppression Therapy/methods , Killer Cells, Natural/immunology , Rats , Rats, Inbred Lew , T-Lymphocytes/immunology , Transplantation, Homologous
5.
Exp Hematol ; 29(3): 339-44, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11274762

ABSTRACT

OBJECTIVE: Organ allografts contain passenger leukocytes that are transferred to the recipient with the transplantation, but their functional relevance to the recipient's immune system is still controversial. MATERIALS AND METHODS: To clarify the functional capacity of passenger leukocytes, we attempted to enhance their effect in rat heart allograft recipients by selective depletion of recipient leukocytes using a monoclonal antibody (mAb) against a recipient-specific allotype of CD45 (RT7(a)). RESULTS: Although antibody treatment of the recipient alone led to profound lymphopenia and reversible myelosuppression, additional transplantation of an major histocompatibility complex-incompatible heart graft from an RT7(b) donor led to lethal aplastic anemia in the recipients. This lethal effect was completely abrogated by postoperative anti-CD3 treatment of the recipient and was partially abrogated or delayed by depletion of passenger leukocytes through additional anti-RT7(b) antibody treatment of the recipient or gamma-irradiation of the graft. CONCLUSIONS: The results suggest a role for both donor and recipient-type T cells for the induction of aplastic anemia in this model. The study shows that, under defined conditions, allogeneic passenger leukocytes in a heart graft can have a profound effect on the recipient's immune system and bone marrow.


Subject(s)
Anemia, Aplastic/etiology , Bone Marrow/pathology , Graft vs Host Reaction , Heart Transplantation/adverse effects , T-Lymphocyte Subsets/transplantation , Transplantation, Homologous/adverse effects , Anemia, Aplastic/immunology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Gamma Rays , Graft Enhancement, Immunologic , Histocompatibility , Immune Tolerance , Isoantibodies/immunology , Leukocyte Common Antigens/immunology , Lymphocyte Depletion , Muromonab-CD3/pharmacology , Muromonab-CD3/therapeutic use , Rats , Rats, Inbred Lew , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/radiation effects
9.
Transplantation ; 72(12): 1898-906, 2001 Dec 27.
Article in English | MEDLINE | ID: mdl-11773886

ABSTRACT

The critical shortage of human donor organs has generated growing interest for porcine to human xenotransplantation. The major immunological barrier to xenotransplantation is the hyperacute rejection (HAR) response that is mediated by preformed xenoreactive antibodies and complement. A promising strategy to control the complement activation, is the expression of human complement regulatory proteins in transgenic animals. We have used the human early cytomegalovirus (CMV) promoter to drive expression of the human complement regulatory protein CD59 (hCD59) in transgenic pigs. A total of eight live transgenic founder animals was born from which five transgenic lines could be established. mRNA analysis and Western blotting revealed high expression of hCD59 in heart, kidney, skeletal muscle, and skin in animals of lines 1 and 5, as well as in the pancreas of four lines. This pattern of expression was confirmed by immunhistological staining. A cell-specific expression in heart and kidney tissue of transgenic lines 1 and 5 was determined. Primary fibroblasts and endothelial cell cultures derived from the aorta of transgenic pigs showed a significantly diminished sensitivity against the challenge with xenoreactive human antibodies and complement whereas non-transgenic control cells were highly susceptible to complement mediated lysis. Ex vivo perfusion of kidneys with pooled human blood revealed a significant protective effect of hCD59 against HAR. The average survival of transgenic kidneys was significantly extended (P<0.05) over nontransgenic controls (207.5+/-54.6 vs. 57.5+/-64.5 min). These data support the concept that hCD59 protects nonprimate cells against human complement mediated lysis and suggest that donor pigs transgenic for hCD59 could play a crucial role in clinical xenotransplantation. Two of five hCD59 transgenic lines showed strong hCD59 expression in several organs relevant for xenotransplantation and a protective effect against HAR. This indicates that the use of the CMV-promoter can facilitate the selection process for optimized transgene expression.


Subject(s)
CD59 Antigens/genetics , Cytomegalovirus/genetics , Gene Expression/physiology , Graft Rejection/prevention & control , Organ Transplantation , Promoter Regions, Genetic/physiology , Swine/genetics , 3T3 Cells , Acute Disease , Animals , Animals, Genetically Modified/genetics , Animals, Genetically Modified/metabolism , Blood Physiological Phenomena , Cell Death/physiology , Cell Membrane/metabolism , Complement System Proteins/physiology , Endothelium, Vascular/cytology , Endothelium, Vascular/physiology , Fibroblasts/physiology , Humans , Immunohistochemistry , Kidney , Mice , Perfusion
10.
Nat Genet ; 26(4): 495-9, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11101853

ABSTRACT

Multiple sclerosis (MS) is the most common demyelinating disease of the central nervous system. It is widely accepted that a dysregulated immune response against brain resident antigens is central to its yet unknown pathogenesis. Although there is evidence that the development of MS has a genetic component, specific genetic factors are largely unknown. Here we investigated the role of a point mutation in the gene (PTPRC) encoding protein-tyrosine phosphatase, receptor-type C (also known as CD45) in the heterozygous state in the development of MS. The nucleotide transition in exon 4 of the gene locus interferes with mRNA splicing and results in altered expression of CD45 isoforms on immune cells. In three of four independent case-control studies, we demonstrated an association of the mutation with MS. We found the PTPRC mutation to be linked to and associated with the disease in three MS nuclear families. In one additional family, we found the same variant CD45 phenotype, with an as-yet-unknown origin, among the members affected with MS. Our findings suggest an association of the mutation in PTPRC with the development of MS in some families.


Subject(s)
Leukocyte Common Antigens/genetics , Multiple Sclerosis/genetics , Multiple Sclerosis/immunology , Point Mutation , Base Sequence , Case-Control Studies , DNA/genetics , DNA Primers/genetics , Exons , Female , Genetic Variation , Heterozygote , Humans , Male , Multiple Sclerosis/enzymology , Pedigree , Phenotype
12.
Hum Immunol ; 61(4): 408-18, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10715518

ABSTRACT

The formation of a trimeric complex, composed of heavy chain (HC), beta(2)-microglobulin (beta(2)m) and antigenic peptide, is generally believed to be a prerequisite for the expression of HLA class I molecules at the cell surface in vivo. Therefore, a possible role in immunological processes for HC/beta(2)m complexes devoid of peptide has not been seriously considered. Using a novel HLA-B*2705-transgenic rat model and monoclonal antibodies that distinguish between structurally different forms of HLA-B27 molecules, we demonstrate here that class I molecules which appear to lack antigenic peptides are expressed in abundance on a variety of cell types in lymphoid organs. These results imply a role for structurally diverse, possibly empty, MHC molecules in physiological T cell selection which has so far not been sufficiently appreciated.


Subject(s)
HLA-B27 Antigen/chemistry , HLA-B27 Antigen/immunology , Amino Acid Sequence , Animals , Animals, Genetically Modified , Antibodies, Monoclonal/metabolism , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Cell Line , Cytokines/pharmacology , HLA-B27 Antigen/genetics , Humans , Lymphocyte Activation/immunology , Molecular Sequence Data , Peptides/chemistry , Peptides/genetics , Peptides/immunology , Rats , Rats, Inbred WKY , Rats, Sprague-Dawley , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Tumor Cells, Cultured
13.
Nat Med ; 5(11): 1292-7, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10545996

ABSTRACT

With an organ transplant, hematopoietic donor cells are transferred to the recipient. To study the relevance of the resulting microchimerism for allograft acceptance, we analyzed a rat model of cyclosporine-induced tolerance for strongly incompatible heart allografts. Using a monoclonal antibody that detects a donor-specific CD45 allotype (RT7a), we selectively depleted donor leukocytes at different times after transplantation (days 0 or 18). Depletion was similarly effective at both times. However, only depletion on day 0 prevented tolerance induction and was associated with severe acute or chronic graft rejection. This indicates that passenger leukocytes have an essential immunomodulatory effect on the induction phase of allograft acceptance.


Subject(s)
Graft Survival/immunology , Heart Transplantation/immunology , Leukocytes/immunology , Transplantation Chimera , Animals , Antibodies, Monoclonal/therapeutic use , Base Sequence , Cytokines/genetics , DNA Primers , Graft Survival/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Inbred Lew , Reverse Transcriptase Polymerase Chain Reaction , Transplantation, Homologous
15.
Eur J Immunol ; 29(5): 1487-95, 1999 05.
Article in English | MEDLINE | ID: mdl-10359102

ABSTRACT

Activated T cells reach the lymph nodes via afferent lymphatics but it is unknown to what extent they also enter them directly via high endothelial venules (HEV). Little is known about the mechanism mediating the proliferation of activated T cells within lymphoid tissues in vivo or the subsequent fate of the progeny. Therefore, we stimulated rat T cells via TCR and CD28 in vitro and after injection identified them in the blood and the HEV of lymphoid organs at several time points. In addition, the proliferation of these cells was studied after entering different lymphoid organs. Our results show that, firstly, activated T cells continuously enter lymph nodes and Peyer's patches directly via HEV. Second, they proliferate within lymphoid organs, the rate significantly depending on the microenvironment. Third, mainly CD8+ progeny are able to leave the tissues and re-enter the blood. Thus, the distribution of activated T cells circulating through the body can be regulated during entry, but also within the tissue by influencing their proliferation and subsequent release.


Subject(s)
CD8-Positive T-Lymphocytes/physiology , Cell Movement/physiology , Lymph Nodes/cytology , Peyer's Patches/cytology , T-Lymphocytes/physiology , Animals , Bromodeoxyuridine/metabolism , CD8-Positive T-Lymphocytes/cytology , Cell Division , Endothelium, Lymphatic , Lymph Nodes/blood supply , Lymphocyte Activation , Rats , Rats, Inbred Lew , T-Lymphocytes/cytology
16.
J Immunol ; 163(1): 40-9, 1999 Jul 01.
Article in English | MEDLINE | ID: mdl-10384097

ABSTRACT

Experimental autoimmune encephalomyelitis (EAE) induced by active immunization with the myelin oligodendrocyte glycoprotein (MOG) is an Ab-mediated, T cell-dependent autoimmune disease that replicates the inflammatory demyelinating pathology of multiple sclerosis. We report that disease susceptibility and severity are determined by MHC and MHC-linked effects on the MOG-specific B cell response that mediate severe clinical EAE in the EAE-resistant Brown Norway (BN) rat. Immunization with the extracellular domain of MOG in CFA induced fulminant clinical disease associated with widespread demyelination and with an inflammatory infiltrate containing large numbers of polymorphonuclear cells and eosinophils within 10 days of immunization. To analyze the effects of the MHC (RT1 system) we compared BN (RT1 n) rats with Lewis (LEW) (RT1 l) and two reciprocal MHC congenic strains, LEW.1N (RT1n) and BN.1L (RT1 l). This comparison revealed that disease severity and clinical course were strongly influenced by the MHC haplotype that modulated the pathogenic MOG-specific autoantibody response. The intra-MHC recombinant congenic strain LEW.1R38 demonstrated that gene loci located both within the centromeric segment of the MHC containing classical class I and class II genes and within the telomeric RT1.M region containing the MOG gene are involved in determining Ab production and disease susceptibility. This study indicates that the current T cell-centered interpretation of MHC-mediated effects on disease susceptibility must be reassessed in multiple sclerosis and other autoimmune diseases in which autoantibody is involved in disease pathogenesis.


Subject(s)
Antigens, Surface/immunology , B-Lymphocytes/immunology , Encephalomyelitis, Autoimmune, Experimental/etiology , Major Histocompatibility Complex/immunology , Myelin-Associated Glycoprotein/immunology , Oligodendroglia/immunology , Adoptive Transfer , Animals , Animals, Congenic , Antigens, Surface/genetics , Autoantibodies/physiology , Disease Susceptibility , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/pathology , Haplotypes , Immunity, Innate , Major Histocompatibility Complex/genetics , Myelin Proteins , Myelin-Associated Glycoprotein/genetics , Myelin-Oligodendrocyte Glycoprotein , Rats , Rats, Inbred BN , Rats, Inbred Lew , Species Specificity , T-Lymphocytes/immunology , T-Lymphocytes/transplantation
18.
Int Immunol ; 11(3): 435-44, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10221655

ABSTRACT

Different rat Tcrb haplotypes express either TCR beta variable segment (Tcrb-V) 8.2l or 8.4a. Both V segments bind the mAb R78 but differ by one conservative substitution (L14V) and clusters of two and four substitutions in the complementarity-determining region (CDR) 2 and CDR4 [hypervariable loop 4 (HV4)]. Independently of MHC alleles numbers of R78+ CD4+ cells are lower in Tcrb-V8.2l-expressing than in Tcrb-V8.4a-expressing strains. Expression of R78+ TCR during T cell development, analysis of backcross populations and generation of a Tcrb congenic strain [LEW.TCRB(AS)] define two mechanisms how Tcrb haplotypes affect the frequency of R78+ cells, one acting prior to thymic selection leading to up to 2-fold higher frequency of Tcrb-V8.4a versus Tcrb-V8.2l in unselected thymocytes and another occurring between the TCRlow and the CD4/CD8 single-positive stage. The latter leads to a 50% reduction of frequency of Tcrb-V8.4a CD8+ cells but not CD4+ cells and does not affect either subset of Tcrb-V8.2l cells. A comparison of rat classical class I MHC (RT1.A) sequences and current models of TCR-MHC-peptide interaction suggests that this reduction in frequency of Tcrb-V8.4a CD8 cells may be a consequence of differential selection of Tcrb-V8.2l versus Tcrb-V8.4a TCR by differential binding of CDR2beta to highly conserved areas of C-terminal parts of the alpha helices of class I MHC molecules.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Major Histocompatibility Complex/immunology , Receptors, Antigen, T-Cell, alpha-beta/genetics , T-Lymphocyte Subsets/immunology , Thymus Gland/immunology , Alleles , Animals , CD4-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/cytology , Cell Differentiation , Crosses, Genetic , Female , Genetic Variation , Haplotypes , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/immunology , Models, Immunological , Rats , Rats, Inbred Strains , T-Lymphocyte Subsets/cytology , Thymus Gland/cytology
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