Theresa Tretter

Contribution of the innate immune system to autoimmune myocarditis: a role for complement

Myocarditis is a principal cause of heart disease among young adults and is often a precursor of heart failure due to dilated cardiomyopathy. We show here that complement is critical for the induction of experimental autoimmune myocarditis and that it acts through complement receptor type 1 (CR1) and type 2 (CR2). We also found a subset of CD44hiCD62Llo T cells that expresses CR1 and CR2 and propose that both receptors are involved in the expression of B and T cell activation markers, T cell proliferation and cytokine production. These findings provide a mechanism by which activated complement, a key product of the innate immune response, modulates the induction of an autoimmune disease.

Induction of CD4 + T-cell anergy and apoptosis by activated human B cells

B cells are well-known mediators of humoral immunity and serve as costimulators in the generation of T cell-mediated responses. In several mouse models, however, it was observed that B cells can also down-regulate immune reactions, suggesting a dual role for B cells. Due to this discrepancy and so far limited data, we directly tested the effects of primary human B cells on activated CD4(+) T helper cells in vitro. We found that under optimal costimulation large, activated CD25(+) B cells but not small CD25(-) B cells induced temporary T-cell anergy, determined by cell division arrest and down-regulation of cytokine production. In addition, large CD25(+) B cells directly induced CD95-independent apoptosis in a subpopulation of activated T cells. Suppression required direct B-T-cell contact and was not transferable from T to T cell, excluding potential involvement of regulatory T cells. Moreover, inhibitory effects involved an IL-2-dependent mechanism, since decreasing concentrations of IL-2 led to a shift from inhibitory toward costimulatory effects triggered by B cells. We conclude that activated CD25(+) B cells are able to costimulate or down-regulate T-cell responses, depending on activation status and environmental conditions that might also influence their pathophysiological impact.

Mimicry of pre-B cell receptor signaling by activation of the tyrosine kinase Blk.

During B lymphoid ontogeny, assembly of the pre–B cell receptor (BCR) is a principal developmental checkpoint at which several Src-related kinases may play redundant roles. Here the Src-related kinase Blk is shown to effect functions associated with the pre-BCR. B lymphoid expression of an active Blk mutant caused proliferation of B progenitor cells and enhanced responsiveness of these cells to interleukin 7. In mice lacking a functional pre-BCR, active Blk supported maturation beyond the pro–B cell stage, suppressed VH to DJH rearrangement, relieved selection for productive heavy chain rearrangement, and stimulated κ rearrangement. These alterations were accompanied by tyrosine phosphorylation of immunoglobulin β and Syk, as well as changes in gene expression consistent with developmental maturation. Thus, sustained activation of Blk induces responses normally associated with the pre-BCR.

CD4+CD25+/highCD127low/- regulatory T cells are enriched in rheumatoid arthritis and osteoarthritis joints—analysis of frequency and phenotype in synovial membrane, synovial fluid and peripheral blood

IntroductionCD4+CD25+/highCD127low/- regulatory T cells (Tregs) play a crucial role in maintaining peripheral tolerance. Data about the frequency of Tregs in rheumatoid arthritis (RA) are contradictory and based on the analysis of peripheral blood (PB) and synovial fluid (SF). Because Tregs exert their anti-inflammatory activity in a contact-dependent manner, the analysis of synovial membrane (SM) is crucial. Published reports regarding this matter are lacking, so we investigated the distribution and phenotype of Tregs in concurrent samples of SM, SF and PB of RA patients in comparison to those of osteoarthritis (OA) patients.MethodsTreg frequency in a total of 40 patients (18 RA and 22 OA) matched for age and sex was assessed by flow cytometry. Functional status was assessed by analysis of cell surface markers representative of activation, memory and regulation.ResultsCD4+ T cells infiltrate the SM to higher frequencies in RA joints than in OA joints (P = 0.0336). In both groups, Tregs accumulate more within the SF and SM than concurrently in PB (P < 0.0001). Relative Treg frequencies were comparable in all compartments of RA and OA, but Treg concentration was significantly higher in the SM of RA patients (P = 0.025). Both PB and SM Tregs displayed a memory phenotype (CD45RO+RA-), but significantly differed in activation status (CD69 and CD62L) and markers associated with Treg function (CD152, CD154, CD274, CD279 and GITR) with only minor differences between RA and OA.ConclusionsTreg enrichment into the joint compartment is not specific to inflammatory arthritis, as we found that it was similarly enriched in OA. RA pathophysiology might not be due to a Treg deficiency, because Treg concentration in SM was significantly higher in RA. Synovial Tregs represent a distinct phenotype and are activated effector memory cells (CD62L-CD69+), whereas peripheral Tregs are resting central memory cells (CD62L+CD69-).

Role of the Cholinergic Antiinflammatory Pathway in Murine Autoimmune Myocarditis

Rationale: This study was performed to gain insights into novel therapeutic approaches for the treatment of autoimmune myocarditis. Objective: Chemical stimulation of the efferent arm of the vagus nerve through activation of nicotinic acetylcholine receptor subtype-7&agr; (&agr;7-nAChR) has been shown to be protective in several models of inflammatory diseases. In the present study, we investigated the potentially protective effect of vagus nerve stimulation on myocarditis. Methods and Results: A/J mice were immunized with cardiac troponin I (TnI) to induce autoimmune myocarditis. Mice were exposed to drinking water that contained nicotine in different concentrations and for different time periods (for 3 days at 12.5 mg/L; 3 days at 125 mg/L; 21 days at 12.5 mg/L; and 21 days at 125 mg/L after first immunization). TnI-immunized mice with no pharmacological treatment showed extensive myocardial inflammation and fibrosis and significantly elevated levels of interleukin-6 and tumor necrosis factor-&agr;. Furthermore, elevated levels of mRNA transcripts of proinflammatory chemokines (monocyte chemoattractant protein-1, macrophage inflammatory protein-1&bgr;, and RANTES) and chemokine receptors (CCR1, CCR2, and CCR5) were found. Oral nicotine administration reduced inflammation within the myocardium, decreased the production of interleukin-6 and tumor necrosis factor-&agr;, and downregulated the expression of monocyte chemoattractant protein-1, macrophage inflammatory protein-1&bgr;, RANTES, CCR1, CCR2, and CCR5. In addition, nicotine treatment resulted in decreased expression of matrix metalloproteinase-14, natriuretic peptide precursor B, tissue inhibitor of metalloproteinase-1, and osteopontin, proteins that are commonly involved in heart failure. Finally, we found that nicotine reduced levels of pSTAT3 (phosphorylated signal transducer and activator of transcription 3) protein expression within the myocardium. Neostigmine treatment did not affect the progression of myocarditis. Conclusions: We showed that activation of the cholinergic antiinflammatory pathway with nicotine reduces inflammation in autoimmune myocarditis. Our results may open new possibilities in the therapeutic management of autoimmune myocarditis.

Complement receptors regulate lipopolysaccharide-induced T-cell stimulation

Complement receptors type 1 and 2 (CR1 (CD35)/CR2 (CD21)) are known to enhance the adaptive immune response. In mice, CR1/CR2 are expressed on B cells, follicular dendritic cells, and activated granulocytes. Recently, we showed that a subset of CD44high and CD62Llow T cells also expresses CR1 and CR2. We now report that CR1/CR2 are detectable on both CD4+ and CD8+ subsets of T cells. Lipopolysaccharide (LPS) from Gram‐negative bacteria causes polyclonal activation of B cells and stimulation of macrophages and other antigen‐presenting cells. We further demonstrate that LPS induced marked up‐regulation of CD25 and CD69 on T cells from CR1/CR2 sufficient (Cr+/+), but significantly lower up‐regulation on T cells from CR1/CR2 deficient (Cr–/–) mice. These findings point to a novel mechanism by which CR1/CR2 modulates the activation of T cells by LPS.

Leflunomide Induces Apoptosis in Fludarabine-Resistant and Clinically Refractory CLL Cells

Purpose: Environmental conditions in lymph node proliferation centers protect chronic lymphocytic leukemia (CLL) cells from apoptotic triggers. This situation can be mimicked by in vitro stimulation with CD40 ligand (CD40L) and interleukin 4 (IL-4). Our study investigates the impact of the drug leflunomide to overcome apoptosis resistance of CLL cells. Experimental Design: CLL cells were stimulated with CD40L and IL-4 and treated with fludarabine and the leflunomide metabolite A771726. Results: Resistance to fludarabine-mediated apoptosis was induced by CD40 activation alone stimulating high levels of BCL-XL and MCL1 protein expression. Apoptosis resistance was further enhanced by a complementary Janus-activated kinase (JAK)/STAT signal induced by IL-4. In contrast, CLL proliferation required both a CD40 and a JAK/STAT signal and could be completely blocked by pan-JAK inhibition. Leflunomide (A771726) antagonized CD40L/IL-4–induced proliferation at very low concentrations (3 μg/mL) reported to inhibit dihydroorotate dehydrogenase. At a concentration of 10 μg/mL, A771726 additionally attenuated STAT3/6 phosphorylation, whereas apoptosis of CD40L/IL-4–activated (“resistant”) CLL cells was achieved with higher concentrations (IC50: 80 μg/mL). Apoptosis was also effectively induced by A771726 in clinically refractory CLL cells with and without a defective p53 pathway. Induction of apoptosis involved inhibition of NF-κB activity and loss of BCL-XL and MCL1 expression. In combination with fludarabine, A771726 synergistically induced apoptosis (IC50: 56 μg/mL). Conclusion: We thus show that A771726 overcomes CD40L/IL-4–mediated resistance to fludarabine in CLL cells of untreated as well as clinically refractory CLL cells. We present a possible novel therapeutic principle for attacking chemoresistant CLL cells. Clin Cancer Res; 18(2); 417–31. ©2011 AACR.

Decreased immunoregulatory activity of B cells derived from patients with systemic lupus erythematosus

Backgroundand objectives Our group has recently generated human B cells with immunoregulatory properties in vitro from peripheral blood (PB) of healthy donors. Upon prestimulation via their B cell receptor (BCR) large, activated CD25+B cells (B25+), but not resting CD25−B-cells (B25−), induced temporary CD4+T cell energy and apoptosis. These results led us to rethink the so far pathogenic role of B cells in autoimmune disease. Our aim was to test the immunoregulatory capabilities of B-cells from patients with systemic lupus erythematosus (SLE), as an autoimmune disease with characteristic B cell involvement. Since Treg defects are reported, at least in advanced stages of disease, it could be suspected that Breg might be affected, as well. Materials and methods Highly purified CD19+B cells and CD4+Th-cells were separated from PBMC by magnetic cell sorting. B cells were prestimulated with SAC (Staphylococcus aureus Cowan I Antigen) for 3d and sorted into highly activated FSChiCD25+ (B25+) and small resting FSCloCD25- (B25-) B cells by cytometric cell sorting. Upon 4d coculture with Th-cells and αCD3+IL-2, T cell proliferation was determined by 3H-TdR incorporation. Experiments were set up in parallel with B cells from healthy donors (ND) and patients with SLE. For better comparison the study additionally included B cells from patients with another autoimmune disease, Wegeners Granulomatosis (WG). Results CD4+T cell proliferation was significantly less inhibited in cocultures with SLE-B25+ compared to cocultures with ND-B25+ (mean 51% vs 35% of proliferation of T cells cultured alone (100%), p<0.01). In cross-over-experiments ND-T cell proliferation decreased below 50% in 22 of 37 cases cocultured with SLE-B25+ but in 34 of 37 cases cocultured with ND-B25+. This effect was independent from T cell origin: SLE-T cell proliferation was similarly reduced below 50% in only 21 of 37 cases with SLE-B cells but in 32 of 37 cases with ND-B cells. Of interest, B25+ cells from patients with WG (n=37) exhibited strong inhibitory effects similar to their normal counterparts. So far, no differences in B cell activation markers, cytokine production or viability were found between ND-, WG- and SLE- B25+-cells. In addition no hints for a correlation between SLE-disease activity, treatment and Breg suppressor-function could be stated. Conclusions B25+ from SLE patients exhibit reduced regulatory capacity towards CD4+T cells in contrast to B25+ from healthy donors or patients with WG, suggesting that suppressive defects of SLE-B cells might be rather disease specific and less representative of autoimmunity in general or chronic inflammation. Future experiments deal with the SLE-B cell specificities interfering with suppressive function and the investigation for parameters to restore it.