The Preliminary Study Of The Effect Of Polyamines On The Binding Of Anti-DNA Antibodies And Its Expression In Systemic Lupus Erythematosus

ObjectiveSystemic lupus erythematosus (SLE) is a prototypic autoimmune disease that caused tissue inflammation and organ injury. The characteristic of SLE is over expression of antibodies to components of the cell nucleus in association with deposition of antigen-antibody immune complex. DNA is one of the most important auto-antigen in SLE that serves as a prominent immunological feature of SLE with diagnostic and prognostic significance. SLE anti-ds DNA antibody primarily binds to DNA conserved phosphodiester backbone while some selectively binds to base determinants. These antibodies vary in specificity and pathogenicity among individuals and species. Polyamines are biogenic small molecular amines that are found abundantly in eukaryotic cell nucleus and prokaryotic cells. They are essential for DNA conformation and gene transcription. To elucidate the specificity of DNA-antibodies interactions as well as the possibility of new treatment to SLE, the effect of polyamines on the binding of anti-DNA antibodies from patients with SLE and normal human subjects (NHS) were tested.MethodsFive sera samples from SLE patients (3 females,2 males, fulfilling the American College of Rheumatology revised classification criteria of SLE in 1997) and five sera samples from normal human subjects (3 females,2 males) as a healthy control were collected. Three kinds of polyamines (spermine, spermidine and putrescine) for their effect on SLE and NHS anti-DNA antibodies binding were test by ELISA. The antibody concentration with polyamine/without polyamine was calculated for the efficiency of polyamine on antibody binding.In addition to examine binding of anti-DNA to calf thymus (CT) DNA as antigen, we also assessed the binding of antibodies to Micrococcus Luteus (MC) DNA. To detect further action of spermine on the binding of anti-DNA differing in specificity, sera were incubated with CT-DNA cellulose first to remove non-specific antibodies, then the antibody level were tested in the presence of spermine. Tetanus toxoid was selected as another antigen and the effect of spermine on anti-tetanus antibody binding of SLE sera was tested to determine the specific function of polyamine on anti-DNA antibody binding. One-way ANOVA and Paired T Test were used for data analysis, P<0.05 was seen as statistical significance.Results1. Spermine at 50-1000μM and spermidine at 100-1000μM can significantly reduce SLE anti-DNA binding to CT DNA (P<0.01); Putrescine has no effect on SLE anti-DNA binding (P>0.05). In the same concentration, the inhibitory activity of spermine is higher than spermidine and putrescine (P<0.01).2. Spermine can not only directly inhibit antibody binding to CT DNA in SLEsera, but also can disassemble pre-formed DNA-antibody immune complexes (P<0.01).3. Spermine does not influence anti-DNA antibody binding to MC DNA in NHS sera (P>0.05).4. Spermine obviously decreased 2 SLE sera S1, S2 anti-DNA antibody binding to MC DNA antigen (P<0.01) but failed to affect the other three SLE sera S3, S4 and S5 anti-DNA antibody binding to MC DNA (P>0.05).5. The anti-MC DNA antibody level was significantly decreased for 5 SLE sera after they absorbed by CT-DNA cellulose (P<0.05). For SLE sera S1, S2, the declined extent of anti-MC antibody was much more than SLE sera S3, S4 and S5 (P<0.01).6. For SLE sera S1, S2, spermine can still inhibit their anti-DNA binding to MC DNA after the sera was absorbed by CT-DNA cellulose (P<0.05), but the extent was less than that absorbed by control cellulose (P<0.01).7. Spermine failed to affect anti-tetanus antibody binding by SLE sera (P>0.05).ConclusionThe binding patterns of antibody to DNA are non-specific binding and specific binding and their ratio in SLE individual is deffer. Spermine can obviously inhibit non-specific anti-DNA antibody binding to DNA antigen in SLE patients and serve as a probe of anti-DNA specificity. The possible mechanism is that polyamines combine with DNA phosphate backbone to alter DNA antigenicity and interfere with antibody and DNA charge-charge interaction. Spermine has no effect on anti-DNA antibody that selectively binds to DNA non-specific determinants and non-DNA antibody. As a small biologic polyamine molecule, spermine can be a new tool for SLE diagnosis and treatment.ObjectivePolyamines (spermine, spermidine and putrescine) are small molecular aliphatic compounds that widely distributed in biologic tissues and cells. They are essential for the regulation of nucleic acid and protein and play a vital role in cell metabolism, proliferation and differentiation. Polyamine’s synthesis and catabolism are homeostasis with precise regulation. Ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) are key enzymes of polyamine synthesis, as well as spermine/spermidine N1-acetyltransferase (SSAT) is the key enzyme of polyamine’s catabolism. Using the technology of high performance lipid chromatography (HPLC), we can depart and detect free polyamines from biologic samples. In the first part of this thesis, we found spermine and spermidine could inhibit some SLE anti-dsDNA antibodies bind to DNA antigen in vitro. In this part we continue tested the mRNA expression levels of ODC, SAMDC and SSAT gene in peripheral blood mononuclear cells (PBMCs) in addition to the concentration of polyamines in plasma of SLE patients and normal human subjects. The aim of this study is to explore the expression status of polyamine in SLE patients and its possible function in SLE occurrence and development.Methods32 SLE patients (28 females,4 males, fulfilling the American college of Rheumatology classification criteria of SLE in 1997) and 30 normal human subjects (26 females,4 males) were enrolled. The ODC, SAMDC and SSAT mRNA levels of PBMCs were determined by Real-Time PCR (RT-PCR); the concentrations of spermine, spermidine and putrescine in plasma were tested by HPLC. Independent T test and Chi square test were used for data analysis. The results of mRNA expression were showed with relative multiples of SLE to NHS group that automatically analyzed by Real-Time PCR software. P<0.05 was seen as statistical significance.Results1. The relative expression of ODC mRNA in SLE group was 0.723, lower than NHS group 1.000. The difference was statistically significant (P<0.05).2. The relative expression of SAMDC mRNA in SLE group 1.101 while the NHS group is 1.000. But the difference has no statistically significant (P>0.05).3. The relative expression of SSAT mRNA in SLE group was 0.664, lower than NHS group 1.000. The difference was statistically significant (P<0.05).4. The plasma putrescine concentration in SLE patients is 1.172ug/ml, lower than the NHS group 1.570ug/ml. The difference was statistically significant (P<0.01).5. The plasma spermidine concentration in SLE patients is 1.303ug/ml while the NHS group is 1.710ug/ml. But the difference has no statistically significant (P>0.05).6. The plasma spermine concentration in SLE patients is 0.471ug/ml, lower than the NHS group 0.603ug/ml. The difference was statistically significant (P<0.05).ConclusionThe expression levels of ODC and SSAT gene mRNA in SLE patients’PBMCs is obviously lower than that in NHS group. The spermine and putrescine concentrations in SLE plasma are significantly lower than NHS plasma, either. Polyamines and its metabolic enzyme genes interact with each other and other factors to keep homeostasis of polyamine. The concentration of polyamine in SLE patients may be related to cell over apoptosis and hormone imbalance.