Eva D. Papadimitraki

Rituximab therapy reduces activated B cells in both the peripheral blood and bone marrow of patients with rheumatoid arthritis: depletion of memory B cells correlates with clinical response

IntroductionBone marrow (BM) is an immunologically privileged site where activated autoantibody-producing B cells may survive for prolonged periods. We investigated the effect of rituximab (anti-CD20 mAb) in peripheral blood (PB) and BM B-cell and T-cell populations in active rheumatoid arthritis (RA) patients.MethodsActive RA patients received rituximab (1,000 mg) on days 1 and 15. PB (n = 11) and BM (n = 8) aspirates were collected at baseline and at 3 months. We assessed B-cell and T-cell populations using triple-color flow cytometry.ResultsRituximab therapy decreased PB (from a mean 2% to 0.9%, P = 0.022) but not BM (from 4.6% to 3.8%, P = 0.273) CD19+ B cells, associated with a significant reduction in the activated CD19+HLA-DR+ subset both in PB (from 55% to 19%, P = 0.007) and in BM (from 68% to 19%, P = 0.007). Response to rituximab was preceded by a significant decrease in PB and BM CD19+CD27+ memory B cells (P = 0.022). These effects were specific to rituximab since anti-TNF therapy did not reduce total or activated B cells. Rituximab therapy did not alter the number of activated CD4+HLA-DR+ and CD4+CD25+ T cells.ConclusionsRituximab therapy preferentially depletes activated CD19+HLA-DR+ B cells in the PB and BM of active RA patients. Clinical response to rituximab is associated with depletion of CD19+CD27+ memory B cells in PB and BM of RA patients.

Genetic, immunologic, and immunohistochemical analysis of the programmed death 1/programmed death ligand 1 pathway in human systemic lupus erythematosus

OBJECTIVE A putative regulatory intronic polymorphism (PD1.3) in the programmed death 1 (PD-1) gene, a negative regulator of T cells involved in peripheral tolerance, is associated with increased risk for systemic lupus erythematosus (SLE). We undertook this study to determine the expression and function of PD-1 in SLE patients. METHODS We genotyped 289 SLE patients and 256 matched healthy controls for PD1.3 by polymerase chain reaction-restriction fragment length polymorphism analysis. Expression of PD-1 and its ligand, PDL-1, was determined in peripheral blood lymphocytes and in renal biopsy samples by flow cytometry and immunohistochemistry. A crosslinker of PD-1 was used to assess its effects on anti-CD3/anti-CD28-induced T cell proliferation and cytokine production. RESULTS SLE patients had an increased frequency of the PD1.3 polymorphism (30.1%, versus 18.4% in controls; P=0.006), with the risk A allele conferring decreased transcriptional activity in transfected Jurkat cells. Patients homozygous for PD1.3-but not patients heterozygous for PD1.3-had reduced basal and induced PD-1 expression on activated CD4+ T cells. In autologous mixed lymphocyte reactions (AMLRs), SLE patients had defective PD-1 induction on activated CD4+ cells; abnormalities were more pronounced among homozygotes. PD-1 was detected within the glomeruli and renal tubules of lupus nephritis patients, while PDL-1 was expressed by the renal tubules of both patients and controls. PD-1 crosslinking suppressed proliferation and cytokine production in both normal and lupus T cells; addition of serum from patients with active SLE significantly ameliorated this effect on proliferation. CONCLUSION SLE patients display aberrant expression and function of PD-1 attributed to both direct and indirect effects. The expression of PD-1/PDL-1 in renal tissue and during AMLRs suggests an important role in regulating peripheral T cell tolerance.

Glomerular expression of toll-like receptor-9 in lupus nephritis but not in normal kidneys: implications for the amplification of the inflammatory response

Toll-like receptors recognising self-derived nucleic acids may participate in the pathogenesis of autoimmune diseases. Following the description of an enhanced population of toll-like receptor-9 (TLR-9) expressing auto-antibody producing B lymphocytes in active lupus, we explored the expression of TLR-9 in the renal tissue of patients with lupus. TLR-9 expression was studied in the kidneys of 12 lupus and 10 control samples from macroscopically unaffected areas of patients with renal adenocarcinoma by immunohistochemistry. A semiquantitative score was assigned separately for tubular, interstitial and glomerular expression. TLR-9 was expressed in the renal tubules and interstitial tissue in both patients with lupus and controls. Six of 12 patients with lupus with proliferative or membranous nephritis – as compared to none of the controls – exhibited both tubulointerstitial and glomerular TLR-9 expression. Biopsies with glomerular TLR-9 expression had a higher activity index (mean ± SD, 6.3 ± 3.5 in the presence of TLR-9 glomerular expression as compared to 1.3 ± 1.8 in its absence, P = 0.015, t-test). This study documents for the first time the up-regulation of TLR-9 within the glomerulus of patients with lupus nephritis. Activation of TLR-9 expressing glomerular cells by endogenous nucleic acids (nucleosomes) may amplify the inflammatory response.

Inhibition of tumour necrosis factor alpha in idiopathic membranous nephropathy: a pilot study

BACKGROUND Tumour necrosis alpha has been implicated in the pathogenesis of autoimmune disorders was to evaluate the safety, tolerability and potential efficacy of the tumour necrosis factor alpha (TNF-alpha) inhibitor, etanercept (ET), in patients with idiopathic membranous nephropathy (MN). METHODS Patients with biopsy-proven MN, nephrotic-range proteinuria and clearance of creatinine 50 ml/min or more were included in the study. Exclusion criteria were treatment with steroids or cyclosporine during the previous 3 months, or cytotoxic agents within 6 months prior to entry. ET was administered subcutaneously, 25 mg twice per week for 3 months. Plasma levels of TNF-alpha, interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-10 (IL-10), soluble intercellular adhesion molecule type 1 (sICAM-1), E-selectin, and the soluble form of tumour necrosis factor receptor-55 (sTNFR-55) were measured on entry and at Months 3, 6, and 9 after commencing therapy. RESULTS Twelve patients were entered in the study (four females/eight males, mean time from diagnosis 8.3 months). The therapy was well tolerated; no infections or other adverse events were recorded by the end of follow-up. Two patients exhibited complete remission of proteinuria for at least 4 years. No significant change was found in the levels of TNF-alpha, IL-1, IL-6, IL-8 and IL-10 during the study. Similarly the levels of E-selectin and sICAM-1 were not significantly altered by therapy. Although we found no change in sTNFR-55 at 3 and 6 months, the levels of sTNFR-55 were found significantly decreased 9 months after therapy (mean difference from baseline: 334 +/- 527 pg/ml, P = 0.028). CONCLUSION Short-term use of ET in a small series of patients reduced sTNFR-55 levels but did not exhibit any significant clinical effect in the majority of patients.

Systemic Lupus Erythematosus: Cytotoxic Agents

Publisher Summary This chapter discusses the use of cytotoxic/immunosuppressive drug therapy in systemic lupus erythematosus (SLE). For each drug under discussion, it provides background information on the mode of action, the pharmacokinetics, clinical use in lupus, and side effects. It also explains both the monitoring of side effects and strategies to minimize them. It focuses on azathioprine, cyclophosphamide, and mycophenolate mofetil as they are the most widely used cytotoxic drugs. Azathioprine (AZA, Imuran) is a cycle-specific antimetabolite that is commonly included in maintenance regimens for lupus nephritis and in regimens against mild-to-moderate SLE. Gastrointestinal complaints such as nausea, vomiting, and diarrhea are the most common side effects of AZA leading approximately 15–30% of patients to the discontinuation of the drug within 6 months. Furthermore, cyclophosphamide (CY) is an alkylating agent whose administration results in cell death that can occur at any stage during the cell cycle. CY depletes both T and B cells, and reduces the production of pathogenic autoantibodies. Reversible alopecia and nausea are the most commonly observed side effects of CY. Finally, mycophenolate mofetil (MMF) is a morpholinoethyl ester and a prodrug of mycophenolic acid (MPA), a potent inhibitor of inosine monophosphate dehydrogenase (IMP-DH), which is indispensible for the de novo synthesis of guanosine nucleotides. Gastrointestinal toxicity including nausea, vomiting, and diarrhea, all alleviated by reducing or splitting the daily dose, are the most common side effects of MMF.

Inflammation and Atherosclerosis

Atherosclerosis is a slowly progressing disorder of large- and medium-sized arteries leading to overt disease when significant narrowing or atherosclerotic plaque rupture occurs. Inflammation plays a key role in atherosclerotic plaque formation, progression and rapture. Both innate and adaptive immune responses are important to disease pathogenesis. Toll-like receptors, NOD-like receptors and the inflammasome are key components of LDL and other atherogenic molecule recognition and internalization and the subsequent interaction with components of the adaptive immune response. The NLPR3 inflammasome acts through the activation of caspase, can be primed by TLR activation and seems to have a crucial role in atherosclerosis and other related disorders such as diabetes, obesity and the metabolic syndrome. However, its experimental inactivation/modification has provided conflicting results in animal models of the disease. Lipid perturbations are also important since they can lead to endothelial cell activation and the augmentation of the inflammatory immune response; even high density lipoproteins may lose their atheroprotective capacities. Chronic inflammatory diseases are characterized by accelerated atherosclerosis: Systemic lupus erythematosus, rheumatoid arthritis, Sjogren syndrome and antiphospholipid syndrome patients display an increased incidence of cardiovascular disease not explained by traditional risk factors.