RESUMO
OBJECTIVE: Complement has both protective and pathogenic functions in lupus due to a balance between its role in the clearance of immune complexes (ICs) and apoptotic cells and its role in inflammation. The classical pathway contributes to IC and apoptotic cell clearance, whereas the alternative pathway is a key mediator of renal inflammation. The aim of this study was to investigate the effect of a new targeted inhibitor of the alternative pathway, CR2-fH, on lupus-like renal disease in MRL/lpr mice. METHODS: Mice were treated with either saline, CR2-fH, CR2-Crry (which inhibits all complement pathways), or soluble CR2 (sCR2; C3d-binding targeting vehicle). Sera were analyzed every 2 weeks for autoantibodies, circulating ICs, and C3. Urinary excretion of albumin was also determined, and kidneys were collected at 23 weeks for histologic evaluation. RESULTS: Treatment with CR2-fH or CR2-Crry improved survival and significantly reduced proteinuria, glomerular C3 deposition, and the level of circulating ICs. CR2-fH, but not CR2-Crry, also significantly reduced glomerulonephritis, expression of serum anti-double-stranded DNA (anti-dsDNA) antibodies, and glomerular IgG and C1q deposition. Interestingly, sCR2 also significantly reduced the levels of anti-dsDNA antibodies and circulating ICs and reduced glomerular deposition of IgG, C1q, and C3, although there was no significant reduction in glomerulonephritis, proteinuria, or mortality. CONCLUSION: Targeted and selective inhibition of the alternative complement pathway is an effective treatment of murine lupus and is more effective than blockade of all pathways. The data demonstrate benefits to leaving the classical/lectin pathways intact and indicate distinct roles for the classical and alternative pathways of complement in disease progression. The sCR2-targeting vehicle contributes to therapeutic activity, possibly via modulation of autoimmunity.
Assuntos
Autoimunidade/efeitos dos fármacos , Inativadores do Complemento/farmacologia , Inativadores do Complemento/uso terapêutico , Via Alternativa do Complemento/efeitos dos fármacos , Nefropatias/tratamento farmacológico , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Animais , Anticorpos Anti-Idiotípicos/sangue , Complexo Antígeno-Anticorpo/sangue , Autoimunidade/fisiologia , Complemento C1q/metabolismo , Complemento C3/metabolismo , Via Alternativa do Complemento/fisiologia , DNA/imunologia , Modelos Animais de Doenças , Feminino , Imunoglobulina G/sangue , Rim/efeitos dos fármacos , Rim/patologia , Nefropatias/etiologia , Nefropatias/imunologia , Lúpus Eritematoso Sistêmico/complicações , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Camundongos Endogâmicos MRL lpr , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
BACKGROUND: Expression of folylpoly-gamma-glutamate synthetase (FPGS) gene is two- to three-fold higher in B-precursor ALL (Bp- ALL) than in T-lineage ALL (T-ALL) and correlates with intracellular accumulation of methotrexate (MTX) polyglutamates and lymphoblast sensitivity to MTX. In this report, we investigated the molecular regulatory mechanisms directing FPGS gene expression in Bp-ALL and T-ALL cells. METHODS: To determine FPGS transcription rate in Bp-ALL and T-ALL we used nuclear run-on assays. 5'-RACE was used to uncover potential regulatory regions involved in the lineage differences. We developed a luciferase reporter gene assay to investigate FPGS promoter/enhancer activity. To further characterize the FPGS proximal promoter, we determined the role of the putative transcription binding sites NFY and E-box on FPGS expression using luciferase reporter gene assays with substitution mutants and EMSA. RESULTS: FPGS transcription initiation rate was 1.6-fold higher in NALM6 vs. CCRF-CEM cells indicating that differences in transcription rate led to the observed lineage differences in FPGS expression between Bp-ALL and T-ALL blasts. Two major transcripts encoding the mitochondrial/cytosolic and cytosolic isoforms were detected in Bp-ALL (NALM6 and REH) whereas in T-ALL (CCRF-CEM) cells only the mitochondrial/cytosolic transcript was detected. In all DNA fragments examined for promoter/enhancer activity, we measured significantly lower luciferase activity in NALM6 vs. CCRF-CEM cells, suggesting the need for additional yet unidentified regulatory elements in Bp-ALL. Finally, we determined that the putative transcription factor binding site NFY, but not E-box, plays a role in FPGS transcription in both Bp- and T-lineage. CONCLUSION: We demonstrated that the minimal FPGS promoter region previously described in CCRF-CEM is not sufficient to effectively drive FPGS transcription in NALM6 cells, suggesting that different regulatory elements are required for FPGS gene expression in Bp-cells. Our data indicate that the control of FPGS expression in human hematopoietic cells is complex and involves lineage-specific differences in regulatory elements, transcription initiation rates, and mRNA processing. Understanding the lineage-specific mechanisms of FPGS expression should lead to improved therapeutic strategies aimed at overcoming MTX resistance or inducing apoptosis in leukemic cells.