Systemic lupus erythematosus (SLE) is an autoimmune disorder of unknown cause characterized by multiple abnormalities in the regulation of the immune response. MAP kinases have been implicated in a broad spectrum of biological responses. Most of previous reports have suggested a defective T-cell activation process in SLE as examined in cultures stimulated with plant-derived mitogens. T cell anergy, as revealed by proliferative unresponsiveness in vitro after stimulation through the T cell receptor (TCR), is associated with defective TCR mediated signal transduction along the PKC/p21ras/p42mapk pathway of T cell activation. The (New Zealand black x New Zealand white [NZB x NZW]) F1 hybrid mouse develops a severe autoimmune disease that closely resembles SLE in human. T cells isolated from F1 hybrid mice will be analyzed by Western blotting assay using anti-MAP and antiphospho-tyrosine antibodies in combination with a functional MAP kinase assay. We then will test whether MAP kinase had any effect on expression of T-cell cytokines genes. Comparing the results between non-transfected F1 hybrid mice T cells and transfected T cells overexpressing MAP kinase, we will find out the mechanisms of defective T-cell activation process in SLE mice could be associated with defective TCR mediated signal transduction along the PKC/p21ras/p42mapk pathway of T cell activation. By examining the amount of transcription factors AP-1, NF-AT, and NF-k B results will indicate in detail that such regulation could be differently mediated through the enhanced DNA binding activity of specific nuclear transcription factors in SLE mice. We can approach in detail to the pathogenesis of SLE by the animal model studies.

The results will prove T cell anergy through the T cell receptor (TCR) is associated with defective TCR mediated signal transduction along the PKC/p21ras/p42mapk pathway of T cell activation in SLE-like mice.

The studies will provide evidences for the role of MAP kinase in the regulation of cytokines gene expression in SLE-like mice.

The results will address in detail that such regulation could be differently mediated through the enhanced DNA binding activity of specific nuclear transcription factors in SLE mice.

There are probably more published studies of immune abnormalities associated with systemic lupus erythematosus(SLE) than with any other single illness. This is due to the striking array of autoantibodies associated with this disorder, leading investigators to study SLE for clues to fundamental principles of self-non-self discrimination (Cohen, P.L.). The mechanisms that underlies pathogenic autoantibodies production are still unclear. The importance of T cells in the induction of lupus has been supported by cell transfer experiments and by amelioration effects of administered anti-CD4 antibodies (Santoro, T.J. et al ). Most of previous reports have suggested a defective T-cell activation process in SLE as examined in cultures stimulated with plant-derived mitogens (Linker-Israeli, et al.). In studies addressing the role of TCR germ line genes, no difference between lupus and normal strains has been detected at the C a , C b , V a , V b loci (Singer, P.A. et al). The expressed T cell receptor repertoire and tolerance-related V b clonal elimination phenomena have also been examined and no defect found (Theofilopoulos, A.N. et al ). No VH germ line of Igh-VH and Vk haplotype associations with autoantibody production and lupus disease have been detected (Kofler, r., et al). Baccalá , R. et al indicated that murine lupus is not associated with defects in central T-cell tolerance as manifested by endogenous superantigen-mediated Vb clonal deletions, and the phenomenon of endogenous superantigen-mediated Vb clonal deletions appears not to be applicable to rats and humans. Their finding suggested that the currently accepted dogma that autoimmunity is the result of breakage in tolerance to unaltered self may be questioned. Therefore, we think the pathogenesis of SLE could be something different inside T cells through defective TCR mediated signal transduction.

Signal transduction in T lymphocytes depends on protein kinases and phosphatases, respectably, to phsophorylate and dephosphorylate signaling intermediates (Taffs, R.E., et al ). Changing profiles of phosphorylated substrates correlated with the progressive series of signaling events that move cells form G0 to G1 and thereafter through the cell cycle. p42mapk is important in mitogenesis and is reversibly phosphorylated during ligation of a number of lymphoid receptors, including CD3, CD2 and mIgM (Casillas, A., et al ). MAP kinases have been implicated in a broad spectrum of biological responses. Two forms of MAP2 kinase have been purified from fibroblasts having Mr.42K (p42mapk) and 44K (p44mapk). Activation of both forms of MAP kinase requires phosphorylation at both tyrosine and threonine residues. Removal of phosphate either from the threonine by phosphatase 2A or from the tyrosine by CD45 results in inactivation of both enzymes. In p42mapk both sites of phosphorylation have been localized : Thr-183 and Tyr-185 (Thomas, G.).

Drewes, G. et al found a protein kinase from brain which specifically induces the Alzheimer-like state in tau protein. The 42kDa protein belongs to the family of mitogen activated protein kinases (MAPKs) and is activated by tyrosine phosphoryl- ation. They proposed that MAP kinase is abnormally active in Alzheimer brain tissue, or that the corresponding phosphatases are abnormally passive, due to a breakdown of the normal regulatory mechanisms. Cross, J.C. et al found the transactivation by Hepatitis B virus X protein dependent on mitogen-activated cellular serine/threonine kinases including protein kinase C and Raf-1. Amaral, M.C. et al thought MAP kinase pathway is important in Kaposi cell growth, because they found oncostatin-M, a 28-kD T cell-derived cytokines, stimulated tyrosine protein phosphorylation in parallel with the activation of p42mapk/ERK-2 in Kaposi's cell. Park, J.H. et al showed that over-expression of MAP1 enhances expression of T-cell interleukin-2 (IL-2), IL-3, and granulocyte-macrophage colony-stimulating factor mRNA. DNA-binding activities of the transcription factors AP1, NF-AT, and NF-kB were specifically increased twofold to fourfold in MAP1-overexpressing clones relative to nontransformed or vector-transformed cells. These results provide direct evidence for the role of MAP kinase in the regulation of cytokines gene expression and indicate that such regulation is likely mediated through the enhanced DNA binding activity of specific nuclear transcription factors.

Rapoport, M. et al recently showed that nonobese diabetic (NOD) mice thymic T cell anergy, as revealed by proliferative unresponsiveness in vitro after stimulation through the T cell receptor(TCR), is associated with defective TCR mediated signal transduction along the PKC/p21ras/ p42mapk pathway of T cell activation. Their findings suggested that a defect in TCR-mediated activation of p21ras may result in reduced tyrosine phosphorylation and activation of p42mapk and p44mapk , which subsequently may inhibit the progression through cell cycle and proliferation of NOD thymocytes. The deficient basal PKC activity in NOD thymic T cells may give rise via the PKC-dependent pathway to decreased p21ras activity in these T cells. Herein, it is an important association exists between a defect(s) in TCR-linked signal transduction and susceptibility to T cell-mediated autoimmune disease. Singh, A.K. also built hypothesis is that multiple defects mediated by cytokines are present in individuals with lupus and that both cytokines production and the response of B cells to cytokines may be defective. These abnormalities could then be a central factor in the etiology of systemic lupus erythematosus. Dauphinee, M.J., et al focused on IL-2, IL-1 and TNF. IL-2 levels in autoimmune mice show an age related decline which correlates with disease activity. Herein we try to find out an important association exists between a defect(s) in TCR-linked signal transduction and susceptibility to T cell-mediated autoimmune disease SLE in animal model.

In animal model, the (New Zealand black x New Zealand white [NZB x NZW]) F1 hybrid mouse develops a severe autoimmune disease that closely resembles SLE in human (Theofilopoulos, A.N., et al ). The defect in Fc-mediated clearance and abnormal complement-mediated clearance were present in NZB/W mice (Meryhew, N.L., et al ). These mice have elevated levels of a number of autoantibodies including antibodies to single-stranded DNA (anti-ssDNA) and anti-thymocyte antibodies, where as the H-2 compatible, non-autoimmune control DBA/2 strain does not have such serum autoantibodies. The level of anti-ssDNA antibodies parallels the development of glomerulonephritis seen in the NZB mice (Laskin, C.A., et al). It offers a very useful animal model to study the mechanisms of SLE.

In this experiment, T cells from the NZB/W F1 hybrid will be purified by monoclonal antibody anti-CD3. After transfecting human MAP1 cDNA expression vector into the F1 hybrid T cells, we will compare the untransfected and transfected F1 T cells IL-2, IL-3 GM-CSF and a actin production by Northern blot. We then check the expression of MAP by Western blot analysis. This data will show whether the defective T cells activation process in SLE mice is through PKC/p21ras/ p42mapk pathway. Furthermore, we will examine the MAP kinase activity in transfected and non-transfected T cells to confirm our results. Then we examine the transcriptional factors associated by MAP kinase pathway from the nuclear extract of two kinds of T cells. Through this way we can understand more detail about the regulation mechanism of T cells in SLE mice. In the future, it could find another way how the autoantibodies could be induced and the role of T cells in autoimmunity diseases. This experiment will provide evidence in support of this notion by demonstrating deficient p21ras activation and p42mapk tyrosine phosphorylation in SLE thymic T cells and reversal of these defects by transfected with MAP kinase expression vector.

Mice. NZB/W F1 mice will be purchased at 4 weeks of age from The Jackson Laboratory (Bar Harbor, ME). Only female F1 mice will be used in this study because of their more rapid onset of autoimmunity (Ishida, H. et al. ).

T Cell isolation. Thymic T cells and purified splenic T cells will be isolated as described (Rapoport, M. et al.) Splenic T cells consist of >95% T cells as estimated by indirect immunofluorescence with the anti-CD3 mAb (Rapoport, M.J. et al).

Plasmid and Transfection. The expression vector pHb -actin-a-Neo, in which expression of the insert Human-MAP1 cDNA is regulated by the human b -actin gene promoter, was graciously provided by Dr. Lee Levitt ( the Division of Hematology, Department of Medicine, Standford University Medical Center, Standford, CA). A concentration of 1 x 107 cells/0.8mL will be suspended in RPMI 1640 containing 10% FCS at room temperature. A total of 30 m g of plasmid DNA will be added to the cell suspension with lipofectin (BRL), incubatedin a humidified incubator at 370C. For transient expression, lipofectin-transfected cells will be transferred to 10 mL of RPMI 1640 containing 10% FCS incubated in a humidified incubator at 370C for 24 hours. The cells then will be stimulated with PHA/PMA for 6 hours and harvested for the b -galacto-sidase assay. As a control for the relative efficiency of transfection, p3'-95-CAT plasmid DNA, in which expression of the chloramphenicol acetyltransferase (CAT) gene is regulated by the promoter fragment of the Chinese hamster ovary (CHO) adenine phosphoribosyl transferase (APRT) gene, a housekeeping gene that is constitutively expressed in all tissues, was cotransfected to T cells. Cell lysates will be also assayed for CAT activity ( Park, J-H. et al).

ELISPOT . The content of the wells will be incubated with 0.1 ml of non-transfected and transfected T cells containing various numbers of SLE mice. Plates will be incubated undisturbed for 3-4 h at 37oC in a CO2 incubator. In third experiment, transfected T cells will be incubated for 5 h at 37oC in a CO2 with various concentration of cycloheximide (Sigma) in assay culture. After incubating IL-2, IL-3 and GM-CSF antibodies, optimal concentrations of AP-conjugated secondary antibodies will be used. Plates will be incubated for 3 h at room temperature. The wells will be exposed to 0.1 ml BCIP/NBT substrate solution and examined for the appearance of blue spots (Czerkinsky, C. et al).

Northern blot analysis. Twenty micrograms of total cellular RNA will be size-fractionated by formaldehyde/agarose gel electrophoresis and transferred to Nytran membrane (Scheichter and Schuell, Keene, NH). The blots will be hybridized with cDNA probes for mouse IL-2, IL-3, GM-CSF, and a actin (American Type Culture Collection, Rockville, MD). All probes will be labeled by random priming. Blots will be finally washed in 0.5X STE( Park, J-H. et al).

Western blot analysis. T cells will be washed twice with cold PBS and resuspended in lysis buffer. The cells will be disrupted by sonication and centrifugation. Ninety micrograms of protein from the cell lysates will be separated on 10% sodium dodecyl sulfate (SDS)-acrylamide gel electrophoresis and transferred to Westran membranes (Schleicher and Schuell, Keene, NH). The ERK-1 production will be analyzed by anti-ERK-1 antiserum Y691 and incubated with I125-labeled protein (Amersham, Arlington Heights, IL). Phosphor Imager (Molecular Dynamics, Sunyvale, CA) will be used to quantitate Northern and Western Blot analyses ( Park, J-H. et al).

Fractionation of cellular lysates and the MAP kinase assay will be performed as described. (Hanekom, C. et al ) The cellular lysates prepared for Western blot analysis will be incubated with DEAE-Sephacel( Pharmacia, Alameda, CA) resin, previously equilibrated in lysis buffer. The MAP kinase assay will be performed by incubating the mixture of 1m g of DEAE-Sephacel elute, MBP(5mg/ml), 50m mol/L[g -P32]-adenosine triphosphate (ATP:3,000 cpm/pmole/L; Amersham). The reaction mixture will be analyzed in SDS-15% polyacrylamide gel ( Park, J-H. et al).

Preparation of nuclear extracts and mobility shift assays. Nuclear extracts will be prepared as described. (Dignam, J.P. et al ) Mobility shift assays will be performed as described. (Park, J-H. et al ) Single-stranded oligonucleotides will be first end-labeled with [g -P32]-ATP. After annealing of complementary oligonucleotides, double-stranded probes will be separated form single-stranded oligonucleotides by 15% polyacrylamide gel electrophoresis (PAGE). The gel slice containing the double-stranded probe will be excised and placed in elution buffer. Nuclear extract will be incubated with 5ng of labeled oligomer. Reaction products will be fractionated by 4% PAGE ( Park, J-H. et al).

In summary, the proposed studies will address the IL-2, IL-3 and GM-CSF RNA and protein expression ; the MAP kinase activity; DNA-binding activity of transcription factors AP1, NF-AT, and NFk B in non-transfected and transfected SLE mice T cells.

Plasticware/glassware $2,000.00

Chemicals/Radionuclides $2,500.00

Antibodies $2,000.00

$6,500.00

Flow cytometry($35)(20 hrs) $ 700.00

New Zealand Black/White F1 mice $1,500.00

Animal care ($0.13/mouse/day) $1,000.00

25% cost of service contract $ 750.00

for gamma counter

$3,950.00

REFERENCES

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Baccalá , R. et al (1992) Lack of evidence for central T-cell tolerance defects in lupus mice and for Vb -deletions endogenous superantigen in rats and humans. Res. Immunol. 143(3):288-90.

Cohen, P.L. (1993) T- and B-cell abnormalities in systemic lupus. J. Invest. Dermatol. 100:69S-72S.

Cross, J.C., et al (1993) Transactivation by hepatitis B virus X protein is promiscuous and dependent on mitogen-activated cellular serine/threonine kinases. Proc. Natl. Acad. Sci. USA. 90:8078-82.

Czerkinsky, C. et al (1988) Anovel two colour ELISPOT assay. J. Immunol. Methods 115:31-37.

Dauphinee, M.J., et al . Interleukin deficiency is a common feature of autoimmune mice. J. Immunol. 127: 2483-87.

Dignam, J.P. et al (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract form isolated mammalian nuclei. Nucleic Acids Res. 11: 1475.

Drewes, G., et al (1992) Mitogen activated protein (MAP) kinase transforms tau protein into an Alzheimer-like state. EMBO J., 11(6):2131-38.

Hanekom, C. et al. (1989) complexing of the CD3 subunit by a monoclonal antibody activates a microtuble-associated protein 2(MAP-2) serine kinase in Jurkat cells. Biochem. J. 262:449.

Ishida, H. et al. (1994) Continuous administration of anti-interleukin 10 antibodies delays onset of autoimmunity in NZB/W F1 mice. J. Exp. Med. 179:305-310.

Kofler, R., et al (1988) Immunoglobulin k chain variable region gene complex organization and Immunoglobulin genes encoding antiDNA antibodies in lupus mice. J. Clin. Invest. 82:852-60.

Laskin, C.A. et al (1986) The regulatory role of NZBT lymphocytes in the production of anti-DNA antibodies in vitro. J. Immunol. 137:1867-73.

Linker-Israeli, et al. (1983) defective production of interleukin 1 and interleukin 2 in patients with systemic lupus erythematosus. J. Immunol. 130:2651.

Meryhew, N.L., et al (1991) Mononuclear phagocyte system dysfunction in murine SLE: Abnormal clearance kinetics precede clinical disease. J. Lab. Clin. Med. 117:181-93.

Park, J-H., et al (1993) Overexpression of mitogen-activated protein kinase(ERK1) enhances T-cell cytokines gene expression: role of AP1, NF-AT, and NF-kB. Blood. 82(8): 2470-77.

Park, J-H. et al. (1993) Transcriptional regulation of interleukin 3 (IL3) in primary human T lymphocytes. Role of AP-1 and octamer-binding proteins in control of IL3 gene expression. J. Biol. Chem. 268:6299.

Rapoport, M.J., et al (1993) Thymic T cell anergy in autoimmune nonobese diabetic mice is mediated by deficient T cell receptor regulation of the pathway of p21ras activation. J. Exp. Med. 177:1221-26.

Santoro, T. J. et al (1988) The contribution of L3T4+ T cells to lymphoproliferation and autoantibody production in MRL-lur/lpr mice. J. Exp. Med. 167:1713-18.

Singer, P.A. et al (1988) T cell receptor, V a haplotype characterization in normal and autoimmune laboratory mouse strains. Proc. Natl. Acad. Sci. USA 85:7729-33.

Singh, A.K. (1992) Cytokines play a central role in the pathogenesis of systemic lupus erythematosus. Medical Hypotheses 39:256-59.

Taffs, R.E., et al (1991) Modulation of cytolytic T-lymphocyte functions by an inhibitor of serine threonine phosphatase, okadaic acid. J.Immunol. 147:722.

Theofilopoulos, A.N. et al (1989) Molecular genetics of murine lupus models. Adv. Immunol. 46:61-109.

Thomas, G. (1992) MAP kinase by any other name smells just as sweet. Cell. 68:3-6.

Kelley et al have found enhanced TNF and IL-1 expression in glomerular macrophages form MRL/lpr mice.

It also remains to be determined whether their findings of decreased activity are restricted to anergic prediabetic NOD T cells or can also be applied to anergic T cells in other autoimmune disease. (Rapoport, M. et al)

The existence of large family is also supported by the fact that two very similar kinases have been identified in budding yeast, which are key components of the mating signal transduction pathway. The gene products , KSS1 FUS3 , share 56% identity with one another and belong to the CDC28/cdc2 family of protein kinases (Thomas, G.).

Daeipour, M. et al used rIL-6 treatment of the human B cell line, AF-10, induced MAP kinase activity. MAP kinase activation in AF-10 cells occurred in parallel with appearance of 42- and 44-kDa tyrosine phosphoproteins. The response was cytokines specific, did not require the presence of extracellular Ca2+, and was minimally affected by the presence of staurosporine.