Interaction of B lymphocytes with neutrophils

GIORDANO M

Mar del Plata

Congreso; LXII Reunión Anual de la Sociedad Argentina de Inmunología; 2014

Chronic lymphocytic leukemia (CLL) is characterized by the progressive accumulation in blood and lymphoid tissues of monoclonal B lymphocytes expressing low levels of surface immunoglobulin (Ig), in most cases IgM. The analysis of the Ig genes in CLL has contributed significantly towards understanding the molecular pathogenesis of the disease. A biased Ig heavy variable (IgVH) gene repertoire suggests a role for antigen(s) in selecting the CLL progenitor cells (1). Recently the study of large number of cases revealed that around 30% of CLL clones carry a restricted set of B cell receptors (stereotyped BCR) that allow the classification of CLL cases into distinct subsets, with particular functional and prognostic features (2). We used nine different IgG recombinant antibodies that expressed stereotyped and non-stereotyped BCRs and found that those belonging to subset #6 recognize the antimicrobial peptide LL37. The precursor of LL37, cathelicidin, is stored in secondary granules of neutrophils and is released and cleaved by proteinase 3 upon activation (3). Because of its cationic nature, LL37 rapidly binds to DNA and is found at high levels on neutrophil extracellular traps (NETs) (4). We evaluated whether recognition of LL37 was able to modify CLL-B cell behavior and unexpectedly we found that not only cells belonging to subset #6, but most CLL clones were protected from spontaneous apoptosis in vitro by LL37, alone or bound to DNA. These results indicate that the effects of LL37 on CLL cells were not related its recognition through the BCR. Of note, culture of CLL cells in the presence of NETs was also able to increase their survival, though to a lesser extent than LL37. Inhibition of apoptosis by the three ligands was accompanied by an increased expression of the activation marker CD86 in CLL cells. There are a number of ways through which LL37 could be inducing these effects. As a cationic peptide, it interacts with heparan sulfate proteoglycans (HSPGs) and might promote the signaling of cell surface receptors including formyl peptide receptor 1 and chemokine receptors (4,5). While growing evidence suggests a direct interaction of LL37 with HSPGs, the exact mechanisms of how LL37 exerts its regulatory functions are yet to be determined. We finally evaluated the capacity of neutrophils from CLL patients to form NETs in vitro when stimulated with PMA. Compared to neutrophils from young and aged-matched healthy donors, neutrophils from CLL patients are prone to form NETs, as indicated by higher levels of DNA and increased elastase activity in supernatants of activated cells. Our findings show that LL37 is one of the antigens that can be recognized by the BCR of a subset of CLL clones and as such may play a role in the initiation and/or progression of the disease. On the other hand, LL37 is able to delay leukemic cell apoptosis and induce activation of most CLL cases by acting on as yet unidentified site. Given that neutrophils from CLL patients are prone to form NETs, the interaction of LL37 with leukemic B cells could occur during their frequent infections.