Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of clonal B-cells in peripheral blood and lymphoid tissues.1 Circulating CLL cells are nondividing B lymphocytes, but a significant fraction of the clone proliferates in lymphoid tissues where they receive a plethora of signals from the microenvironment that promote their survival and expansion.2 Cathelicidins are a family of proteins with antibacterial functions mainly expressed by neutrophils, macrophages and epithelial cells.3 In humans, the only member of this family, hCAP18, is encoded by the gene CAMP. The cleavage of hCAP18 generates the antimicrobial peptide LL-37, which has been recently implicated in the promotion of tumor growth, through direct stimulation of malignant cells, initiation of angiogenesis and recruitment of immune cells.4 In this study, we investigated the role of hCAP18/LL-37 in CLL.
Clinical features of analyzed patients are shown in the Online Supplementary Table S1. By gene expression analysis, we found that CAMP is up-regulated in CLL cells compared to B cells from age-matched healthy donors (HD) (Online Supplementary Figure S1), and this was confirmed by qRT-PCR (Figure 1A). Interestingly, when samples were discriminated according to their immunoglobulin heavy chain variable region (IGHV) mutation status, those with unmutated IGHV (poor clinical outcome, UCLL) showed significantly higher levels than those with mutated IGHV (better outcome, M-CLL). Similarly, leukemic cells from patients in Rai III/IV stages expressed higher levels of hCAP18 mRNA compared to indolent Rai 0 patients (Figure 1B), suggesting that transcription of CAMP is related to an aggressive clinical phenotype. Nevertheless, flow cytometry analysis, using polymorphonuclear cells (PMN) as a positive control, did not reveal hCAP-18/LL-37 protein expression in circulating CLL cells or B lymphocytes from healthy donors (Online Supplementary Figure S2).
Given that CLL cells in lymphoid tissues possess a distinct phenotype, with enhanced expression of anti-apoptotic and activation markers compared to circulating cells;5 we looked for hCAP18/LL-37 in bone marrow biopsies. Figure 1C shows that a proportion of leukemic cells (CD20+) and almost all myeloid cells (CD68+) cells expressed hCAP18/LL-37. These results suggest that signals from the microenvironment promote the expression of hCAP-18/LL-37 protein in CLL cells. To assess this possibility we stimulated circulating CLL cells with different well-known stimuli, such as CD40L, CpG, immobilized anti-IgM, interleukin (IL)-4 (IL-4) and IL-15. Results show that these stimuli up-regulated the transcription of CAMP (Figure 1D) and, more importantly, those stimuli that were most effective in increasing hCAP18 mRNA, i.e., CpG+IL15 and CD40L+IL4, induce the expression of hCAP18/LL-37 protein (Figure 1E). We also observed a significant though moderate correlation between mRNA and protein levels of hCAP18/LL-37 after cellular activation (Online Supplementary Figure S3). Compared to neutrophils, the intracellular expression of hCAP18/LL-37 in activated CLL was modest. However it should be taken into account that hCAP18/LL-37 is stored in neutrophils granules at very high concentrations (0.6 mg/106 cells),6 while it appears to be released to supernatant by activated CLL cells (Figure 1F).
hCAP18/LL-37 has been shown to participate as a tumor promoting factor in breast and lung cancers and in pancreatic ductal adenocarcinoma.4,7 In those settings, hCAP18/LL-37 acts mainly as a growth factor inducing tumor cell activation, survival and proliferation. Since CLL cells proliferate minimally in vitro, even upon strong stimulation, we assessed if LL-37 could induce leukemic cell activation and inhibit spontaneous and drug-induced apoptosis. We found that incubation of CLL cells with LL-37 at 5 or 10 mM did not increase the expression of activation markers (not shown), but significantly delayed spontaneous apoptosis of leukemic cells as evaluated by binding of Annexin V (Figure 2A-B). These concentrations are found at sites of inflammation, like bronchoalveolar lavage fluid from infants with pulmonary infections8 or psoriatic skin lesions.9 Of note, LL-37 not only inhibited spontaneous CLL apoptosis but also interfered with that induced by ABT-199 or fludarabine, agents currently employed for CLL treatment1 (Figure 2CD and Online Supplementary Figures 4-5). In addition to AnnexinV staining, inhibition of apoptosis by LL-37 was corroborated analyzing caspase 3 cleavage (Figure 2E) and BCL-2 expression (Online Supplementary Figure S5B). Because delay of neutrophil apoptosis by LL-37 has been reported to depend on two different receptors: FPRL1 and P2X7,10 we tested if WRW4 (FPRL1 antagonist) or KN-62 (P2X7 inhibitor) could counteract the protective effect of LL-37. Figure 3A shows that none of these agents interfered with LL-37 activity indicating that neither FPRL1 nor P2X7 were involved.
LL-37 can also interact with CXCR4,11 the receptor for the chemokine CXCL12, which plays an important role in CLL by promoting the migration of leukemic cells to lymphoid tissues and improving their survival.12 Therefore, we determined if CXCR4 was involved in the anti-apoptotic pathway triggered by LL-37. We found that an antibody directed to CXCR4 that blocks CXCL12 engagement impaired LL-37 protection (Figure 3B). Furthermore, confocal microscopy shows co-localization of LL-37 and CXCR4 on the cell membrane indicating that both molecules are in the same patches on CLL surface (Figure 3C). Finally, we evaluated CXCR4 endocytosis since this is a mandatory step for chemokine receptors upon binding cognate chemokine, in this case CXCL12. The interaction of CXCR4 with LL-37 at 37ºC led to downregulation of CXCR4 from the CLL cell surface (Figure 3D). Altogether, these results suggest that CXCR4 could be acting as a LL-37 receptor on CLL cells.
Since it has previously been reported that LL-37 enhances the chemotactic responsiveness of hematopoietic progenitors to low doses of CXCL12,11 we evaluated if this was also true for CLL cells. To address this, we used transwell chambers and assessed leukemic cell migration towards a suboptimal concentration of CXCL12 (25 ng/mL), with or without LL-37 (5 mM). As was the case for hematopoietic progenitors, LL-37 increased the migratory response of CLL cells to CXCL12. Of note, LL-37 did not induce chemotaxis of CLL cells by itself, as demonstrated in neutrophils acting through FPRL110 and in keratinocytes through transactivation of the epidermal growth factor receptor13 (Figure 3E).
Accumulating evidence supports pro- or anti-tumorigenic roles for hCAP18/LL-37 in various types of human cancer, e.g., pro-tumor in ovarian, lung and breast cancer and in malignant melanoma, and anti-tumor in colon and gastric tumors.4 In agreement with its inflammatory and pro-angiogenic activity, hCAP-18/LL-37 expression in most solid tumors correlates with increased leukocyte infiltration and microvessel density. In breast and lung cancers, exogenous LL-37 promotes cell proliferation and metastasis,4 but it induces G0/G1 cell cycle arrest in gastric cancer cells, suggesting that the effect of hCAP18/LL- 37 depends on the tissue origin of the tumor. Moreover, it is important to consider that hCAP18/LL-37 can also be expressed by stromal and myeloid cells in the microenvironment. In fact, malignant cells are able to promote the expression of hCAP18/LL-37 by tumor associated macrophages as occurs in pancreatic ductal adenocarcinomas where the secreted peptide contributes to tumor progression.7 Similarly, in a murine model of prostatic cancer, overexpression of CAMP in tumor cells favors the differentiation of early myeloid progenitors into protumorigenic M2 macrophages.14
Here we show that, unlike normal B lymphocytes, leukemic B cells from CLL patients express hCAP18 mRNA and that this expression associates with poor prognosis and disease stage. Interestingly, we found that circulating CLL cells can produce and release hCAP18/LL-37 protein upon in vitro activation with microenvironmental stimuli and this observation was supported by bone marrow immunohistochemistry. In vitro, exogenous LL-37 delays CLL cell apoptosis, both spontaneous and apoptosis induced by chemotherapeutic agents. As an antimicrobial peptide, the main function of LL-37 is to induce bacterial lysis via the formation of transmembrane pores.15 Cellular membranes associated with cholesterol, such as those from human cells, are resistant to LL-37 toxicity unless supraphysiological concentrations are used. By contrast, LL-37 has antiapoptotic effects in a variety of cells including neutrophils through FPRL1 and P2X7.10 We have shown that these two receptors are not involved in CLL cell protection by LL-37, but rather this depends on CXCR4, a key receptor for CLL cell survival and traffic. Of note, LL-37 was able to enhance leukemic cell migration towards suboptimal concentrations of CXCL12.
In conclusion, our results suggest that hCAP18/LL-37 may have an active role in the CLL-tumor microenvironment by increasing leukemic clone retention and survival in lymphoid tissues.
this work was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica, Argentina.
The authors would like to thank María Tejeda, Romina Pagano and Federico Fuentes for technical assistance. We are grateful for the participation of the patients and control subjects in this study.
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