Chamilly Evaldsson

Lymphoblastoid cell line with B1 cell characteristics established from a chronic lymphocytic leukemia clone by in vitro EBV infection

Chronic lymphocytic leukemia (CLL) cells express the receptor for Epstein-Barr virus (EBV) and can be infected in vitro. Infected cells do not express the growth-promoting set of EBV-encoded genes and therefore they do not yield LCLs, in most experiments. With exceptional clones, lines were obtained however. We describe a new line, HG3, established by in vitro EBV-infection from an IGHV1–2 unmutated CLL patient clone. All cells expressed EBNA-2 and LMP-1, the EBV-encoded genes pivotal for transformation. The karyotype, FISH cytogenetics and SNP-array profile of the line and the patients ex vivo clone showed biallelic 13q14 deletions with genomic loss of DLEU7, miR15a/miR16–1, the two micro-RNAs that are deleted in 50% of CLL cases. Further features of CLL cells were: expression of CD5/CD20/CD27/CD43 and release of IgM natural antibodies reacting with oxLDL-like epitopes on apoptotic cells (cf. stereotyped subset-1). Comparison with two LCLs established from normal B cells showed 32 genes expressed at higher levels (> 2-fold). Among these were LHX2 and LILRA. These genes may play a role in the development of the disease. LHX2 expression was shown in self-renewing multipotent hematopoietic stem cells, and LILRA4 codes for a receptor for bone marrow stromal cell antigen-2 that contributes to B cell development. Twenty-four genes were expressed at lower levels, among these PARD3 that is essential for asymmetric cell division. These genes may contribute to establish precursors of CLL clones by regulation of cellular phenotype in the hematopoietic compartment. Expression of CD5/CD20/CD27/CD43 and spontaneous production of natural antibodies may identify the CLL cell as a self-renewing B1 lymphocyte.

Silenced B-cell receptor response to autoantigen in a poor-prognostic subset of chronic lymphocytic leukemia

Chronic lymphocytic leukemia B cells express auto/xeno antigen-reactive antibodies that bind to self-epitopes and resemble natural IgM antibodies in their repertoire. One of the antigenic structures recognized is oxidation-induced malonedialdehyde that is present on low-density lipoprotein, apoptotic blebs, and on certain microbes. The poor-prognostic stereotyped subset #1 (Clan I IGHV genes-IGKV1(D)-39) express IgM B-cell receptors that bind oxidized low-density lipoprotein. In this study, we have used for the first time this authentic cognate antigen for analysis of downstream B-cell receptor-signal transduction events, since it is more faithful to B-cell physiology than anti-IgM. Multivalent oxidized low-density lipoprotein showed specific binding to subset #1 IgM/IgD B-cell receptors, whereas native low-density lipoprotein did not. The antigen binding induced prompt receptor clustering followed by internalization. However, the receptor-signal transduction was silenced, revealing no Ca2+ mobilization or cell-cycle entry, while phosphorylated extracellular-regulated kinase 1/2 basal levels were high and could not be elevated further by oxidized low-density lipoprotein. Interestingly, B-cell receptor responsiveness was recovered after 48-h culture in the absence of antigen in half of the cases. Toll-like receptor 9-ligand was found to breach the B-cell receptor-signaling incompetence in 5 of 12 cases pointing to intra-subset heterogeneity. Altogether, this study supports B-cell receptor unresponsiveness to cognate self-antigen on its own in poor-prognostic subset #1 chronic lymphocytic leukemia, indicating that these cells proliferate by other mechanisms that may override B-cell receptor silencing brought about in a context of self-tolerance/anergy. These novel findings have implications for the understanding of chronic lymphocytic leukemia pathobiology and therapy.

4-Thiouridine induces dose-dependent reduction of oedema, leucocyte influx and tumour necrosis factor in lung inflammation

Recent reports demonstrate a role for nucleotides as inflammatory modulators. Uridine, for example, reduces oedema formation and leucocyte infiltration in a Sephadex‐induced lung inflammation model. Tumour necrosis factor (TNF) concentration was also reduced. Previous in vivo observations indicated that 4‐thiouridine might have similar effects on leucocyte infiltration and TNF release. The aim of this study was thus to investigate the effects of 4‐thiouridine in greater detail. We used a Sephadex‐induced acute lung inflammation model in Sprague–Dawley rats. The dextran beads were instilled intratracheally into the lungs, which were excised and examined after 24 h. Sephadex alone led to massive oedema formation and infiltration of macrophages, neutrophils and eosinophils. Microgranulomas with giant cell formations were clearly visible around the partially degraded beads. A significant increase in bronchoalveolar lavage fluid (BALF) content of TNF and leukotrienes was also seen. 4‐Thiouridine co‐administration affected all variables investigated in this model, i.e. oedema, microscopic and macroscopic appearance of lung tissue, total leucocyte and differential leucocyte counts in BALF, TNF and leukotrienes C4 (LTC4), LTD4 and LTE4 in BALF, indicating a reproducible anti‐inflammatory effect. In conclusion, we have demonstrated that 4‐thiouridine has anti‐inflammatory effects similar to those of uridine. To our knowledge, this is the first demonstration of pharmacological 4‐thiouridine effects in vivo. The results suggest nucleoside/nucleotide involvement in inflammatory processes, warranting further studies on nucleoside analogues as attractive new alternatives in the treatment of inflammatory diseases.

Isomaltitol Exacerbates Neutrophilia but Reduces Eosinophilia: New Insights into the Sephadex Model of Lung Inflammation

Background: We have previously examined isomaltitol in an in vitro static adhesion assay and were interested in investigating whether the potentially anti-inflammatory effects observed there could be relevant in vivo. The Sephadex-induced lung inflammation model was considered a suitablemodel due to the significant changes in global inflammatory endpoints seen upon provocation with Sephadex. Methods: Male Sprague-Dawley rats were instilled intratracheally with Sephadex (5 mg/ml), vehicle (0.9% NaCl), isomaltitol (50 mg/ml) or a combination of isomaltitol and Sephadex. After 24 h, the lungs were weighed to measure edema and preserved for histology. Bronchoalveolar lavage fluid was used for analysis of tumor necrosis factor, cysteinyl leukotrienes, and differential and total leukocyte counts. Results: Differential counts showed that isomaltitol increased the neutrophil component while decreasing the eosinophilia, thus asserting a modulatory role on the usually eosinophil-dominated Sephadex-induced cell profile. Isomaltitol alone also increased edema and cysteinyl leukotrienes, and generally aggravated total inflammation in combination with Sephadex. The mechanisms were not investigated in this study, but effects could relate to a combination of isomaltitol’s osmotic and structure-specific properties. Conclusion: Our results show that isomaltitol can modulate the inflammatory response induced by Sephadex instillation in addition to having proinflammatory effects on its own, and may therefore provide new insights into the mechanisms of this widely used animal model. Sugar alcohols similar to isomaltitol have already been used to aid mucus clearance in cystic fibrosis patients, and it is possible that isomaltitol could also be used for this purpose.

Soluble Fas ligand is associated with natural killer cell dynamics in coronary artery disease

OBJECTIVE Apoptosis of natural killer (NK) cells is increased in patients with coronary artery disease (CAD) and may explain why NK cell levels are altered in these patients. Soluble forms of Fas and Fas ligand (L) are considered as markers of apoptosis. Here, we investigated whether plasma levels of Fas and FasL were associated with NK cell apoptosis and NK cell levels in CAD patients. METHODS Fas and FasL in plasma were determined by ELISA in 2 cohorts of CAD patients; one longitudinal study measuring circulating NK cells and apoptotic NK cells by flow cytometry 1 day, 3 months and 12 months after a coronary event and one cross-sectional study measuring NK cell apoptosis ex vivo. Both studies included matched healthy controls. Fas and FasL were also determined in supernatants from NK cells undergoing cytokine-induced apoptosis in cell culture. RESULTS In the 12-month longitudinal study, plasma FasL increased by 15% (p<0.001) and NK cell levels by 31% (p<0.05) while plasma Fas did not change. Plasma FasL and NK cell levels were significantly related at 3 months and 12 months, r=0.40, p<0.01. Furthermore, plasma FasL, but not plasma Fas, correlated with NK cell apoptosis ex vivo in CAD patients, r=0.54, p<0.05. In vitro, cytokine-induced apoptosis of NK cells resulted in abundant release of FasL. CONCLUSION In CAD patients, FasL in plasma is associated with both apoptotic susceptibility of NK cells and dynamic changes in circulating NK cells. NK cells are also themselves a potential source of soluble FasL. Our findings link NK cell status to a soluble marker with possible atheroprotective effects thereby supporting a beneficial role of NK cells in CAD.