CD40 Expression and its Association with Low-grade Inflammation in a Greek Population of Type 1 Diabetic Juveniles: Evidence for Differences in CD40 mRNA Isoforms Expressed by Peripheral Blood Mononuclear Cells

Abstract

Background: CD40 signalling has been associated with the pathogenesis of autoimmune diseases and diseases with low-grade chronic inflammation.

Objective: To investigate, early in the course of type 1 diabetes (T1DM) patients, the expression of CD40 system components, as well as to explore the association of plasma and urine concentrations of CD40 with known inflammatory markers in T1DM.

Methods: Plasma, urine and peripheral blood mononuclear cells (PBMCs) from 70 T1DM patients without clinically detected chronic complications and 40 healthy controls (HCs) were examined using ELISA, western-blot, semi-quantitative RT-PCR and DNA-sequencing.

Results: Patients had significantly higher plasma soluble CD40 (sCD40) levels associated with higher Interleukin-6 (IL-6), matrix metalloproteinase-9 (MMP-9) and CRP levels compared with healthy controls. This difference was also evident between poorly and well-controlled diabetic patients. The elevated plasma sCD40 levels do not appear to be due to diminished renal excretion since sCD40 concentrations in the urine were also elevated, suggesting an increased CD40 production. An upregulation of PBMCs’ CD40 was evident in T1DM patients associated with higher sCD40, IL-6 and CRP levels. Furthermore, the main CD40 isoform (isoform-I) was solely expressed in poorly controlled diabetics’ PBMCs, who also demonstrated cellular CD40 upregulation, higher plasma CD40, CRP, IL-6 and MMP-9 levels compared with the well-controlled diabetics and the control group, who co-expressed type I and II isoforms.

Conclusions: Homeostatic dysregulation of CD40 and its association with inflammatory markers in T1DM patients, especially in those with poor glycaemic control, implies a pathophysiological role of CD40 in the low-grade inflammatory process in T1DM.

Key words

type 1 diabetes (T1DM) - CD40 - inflammation - PBMC - isoforms - IL-6

References

• 1 Abrahamian H, Endler G, Exner M. et al . Association of low-grade inflammation with nephropathy in type 2 diabetic patients: role of elevated CRP-levels and 2 different gene-polymorphisms of proinflammatory cytokines.  Exp Clin Endocrinol Diabetes. 2007;  115 38-41
• 2 Balasa B, Krahl T, Patstone G. et al . CD40 ligand-CD40 interactions are necessary for the initiation of insulitis and diabetes in nonobese diabetic mice.  J Immunol. 1997;  159 4620-4627
• 3 Basok A, Shnaider A, Man L. et al . CD40 is expressed on human peritoneal mesothelial cells and upregulates the production of interleukin-15 and RANTES.  J Am Soc Nephrol. 2001;  12 695-702
• 4 Braesch-Andersen S, Paulie S, Koho H. et al . Biochemical characteristics and partial amino acid sequence of the receptor-like human B cell and carcinoma antigen CDw40.  J Immunol. 1989;  142 562-567
• 5 Chase HP, Cooper S, Osberg I. et al . Elevated C-reactive protein levels in the development of type 1 diabetes.  Diabetes. 2004;  53 2569-2573
• 6 Chen Y, Chen J, Xiong Y. et al . Internalization of CD40 regulates its signal transduction in vascular endothelial cells.  Biochem Biophys Res Commun. 2006;  345 106-117
• 7 Chiarelli F, Giannini C, Verrotti A. et al . Increased concentrations of soluble CD40 ligand may help to identify type 1 diabetic adolescents and young adults at risk for developing persistent microalbuminuria.  Diabetes Metab Res Rev. 2008;  24 570-576
• 8 Cifarelli V, Libman IM, Deluca A. et al . Increased Expression of Monocyte CD11b (Mac-1) in Overweight Recent-Onset Type 1 Diabetic Children.  Rev Diabet Stud. 2007;  4 112-117
• 9 Contin C, Pitard V, Delmas Y. et al . Potential role of soluble CD40 in the humoral immune response impairment of uraemic patients.  Immunology. 2003;  110 131-140
• 10 Contin C, Pitard V, Itai T. et al . Membrane-anchored CD40 is processed by the tumor necrosis factor-alpha-converting enzyme. Implications for CD40 signaling.  J Biol Chem. 2003;  278 32801-32809
• 11 Dasu MR, Devaraj S, Jialal I. High glucose induces IL-1beta expression in human monocytes: mechanistic insights.  Am J Physiol Endocrinol Metab. 2007;  293 E337-346
• 12 Devaraj S, Glaser N, Griffen S. et al . Increased monocytic activity and biomarkers of inflammation in patients with type 1 diabetes.  Diabetes. 2006;  55 774-779
• 13 Gerritse K, Laman JD, Noelle RJ. et al . CD40-CD40 ligand interactions in experimental allergic encephalomyelitis and multiple sclerosis.  Proc Natl Acad Sci USA. 1996;  93 2499-2504
• 14 Henn V, Slupsky JR, Grafe M. et al . CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells.  Nature. 1998;  391 591-594
• 15 Holler N, Kataoka T, Bodmer JL. et al . Development of improved soluble inhibitors of FasL and CD40L based on oligomerized receptors.  J Immunol Methods. 2000;  237 159-173
• 16 Jinchuan Y, Zonggui W, Jinming C. et al . Upregulation of CD40  CD40 ligand system in patients with diabetes mellitus.  Clin Chim Acta. 2004;  339 85-90
• 17 Kamper EF, Kopeikina LT, Karajiannis CH. et al . Tetranectin, soluble P-selectin and VCAM-1 in the plasma of children with insulin-dependent diabetes mellitus.  Fibrinolysis Proteolysis. 1999;  13 148-153
• 18 Karavitaki N, Lembessis P, Tzanela M. et al . Molecular staging using qualitative RT-PCR analysis detecting thyreoglobulin mRNA in the peripheral blood of patients with differentiated thyroid cancer after therapy.  Anticancer Res. 2005;  25 3135-3142
• 19 Kenyon NS, Chatzipetrou M, Masetti M. et al . Long-term survival and function of intrahepatic islet allografts in rhesus monkeys treated with humanized anti-CD154.  Proc Natl Acad Sci USA. 1999;  96 8132-8137
• 20 Khuu HM, Robinson CA, Goolsby K. et al . Evaluation of a fully automated high-performance liquid chromatography assay for hemoglobin A1c.  Arch Pathol Lab Med. 1999;  123 763-767
• 21 Kreuzer KA, Lass U, Landt O. et al . Highly sensitive and specific fluorescence reverse transcription-PCR assay for the pseudogene-free detection of beta-actin transcripts as quantitative reference.  Clin Chem. 1999;  45 297-300
• 22 Laman JD, de Smet BJ, Schoneveld A. et al . CD40-CD40L interactions in atherosclerosis.  Immunol Today. 1997;  18 272-277
• 23 MacDonald KP, Nishioka Y, Lipsky PE. et al . Functional CD40 ligand is expressed by T cells in rheumatoid arthritis.  J Clin Invest. 1997;  100 2404-2414
• 24 Mach F, Schonbeck U, Sukhova GK. et al . Functional CD40 ligand is expressed on human vascular endothelial cells, smooth muscle cells, and macrophages: implications for CD40-CD40 ligand signaling in atherosclerosis.  Proc Natl Acad Sci USA. 1997;  94 1931-1936
• 25 Mitropapas G, Nezos A, Halapas A. et al . Molecular detection of tyrosinase transcripts in peripheral blood from patients with malignant melanoma: correlation of PCR sensitivity threshold with clinical and pathologic disease characteristics.  Clin Chem Lab Med. 2006;  44 1403-1409
• 26 Mylona-Karayanni C, Gourgiotis D, Bossios A. et al . Oxidative stress and adhesion molecules in children with type 1 diabetes mellitus: a possible link.  Pediatr Diabetes. 2006;  7 51-59
• 27 Nishikawa T, Edelstein D, Du XL. et al . Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage.  Nature. 2000;  404 787-790
• 28 Parker DC, Greiner DL, Phillips NE. et al . Survival of mouse pancreatic islet allografts in recipients treated with allogeneic small lymphocytes and antibody to CD40 ligand.  Proc Natl Acad Sci USA. 1995;  92 9560-9564
• 29 Rasband WS. Image J. In: National Institutes of Health. Bethesda, Maryland, USA 1997–2004
• 30 Schonbeck U, Libby P. The CD40/CD154 receptor/ligand dyad.  Cell Mol Life Sci. 2001;  58 4-43
• 31 Schonbeck U, Mach F, Libby P. Generation of biologically active IL-1 beta by matrix metalloproteinases: a novel caspase-1-independent pathway of IL-1 beta processing.  J Immunol. 1998;  161 3340-3346
• 32 Schwabe RF, Engelmann H, Hess S. et al . Soluble CD40 in the serum of healthy donors, patients with chronic renal failure, haemodialysis and chronic ambulatory peritoneal dialysis (CAPD) patients.  Clin Exp Immunol. 1999;  117 153-158
• 33 Shanmugam N, Reddy MA, Guha M. et al . High glucose-induced expression of proinflammatory cytokine and chemokine genes in monocytic cells.  Diabetes. 2003;  52 1256-1264
• 34 Sharma MD, Leite de Moraes M, Zavala F. et al . Induction and inhibition of CD40-CD40 ligand interactions: a new strategy underlying host-virus relationships.  J Immunol. 1998;  161 5357-5365
• 35 Shiau MY, Tsai ST, Tsai KJ. et al . Increased circulatory MMP-2 and MMP-9 levels and activities in patients with type 1 diabetes mellitus.  Mt Sinai J Med. 2006;  73 1024-1028
• 36 Sowers JR. Diabetes mellitus and cardiovascular disease in women.  Arch Intern Med. 1998;  158 617-621
• 37 Stamm S, Ben-Ari S, Rafalska I. et al . Function of alternative splicing.  Gene. 2005;  344 1-20
• 38 Targher G, Zenari L, Bertolini L. et al . Elevated levels of interleukin-6 in young adults with type 1 diabetes without clinical evidence of microvascular and macrovascular complications.  Diabetes Care. 2001;  24 956 957
• 39 Tone M, Tone Y, Fairchild PJ. et al . Regulation of CD40 function by its isoforms generated through alternative splicing.  Proc Natl Acad Sci USA. 2001;  98 1751-1756
• 40 Town T, Tan J, Mullan M. et al . CD40 signaling and Alzheimer's disease pathogenesis.  Neurochem Int. 2001;  39 371-380
• 41 van Kooten C, Banchereau J. CD40-CD40 ligand.  J Leukoc Biol. 2000;  67 2-17
• 42 van Kooten C, Gaillard C, Galizzi JP. et al . B cells regulate expression of CD40 ligand on activated T cells by lowering the mRNA level and through the release of soluble CD40.  Eur J Immunol. 1994;  24 787-792
• 43 Yellin MJ, Winikoff S, Fortune SM. et al . Ligation of CD40 on fibroblasts induces CD54 (ICAM-1) and CD106 (VCAM-1) up-regulation and IL-6 production and proliferation.  J Leukoc Biol. 1995;  58 209-216

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