Abstract
It has been shown that β-(1 → 3)-(1 → 4)-glucans (BG34) from barley and oats can trigger recognition and internalization by murine and human macrophages. Increasing evidence has suggested that macrophage recognition and internalization of BG34 are dramatically affected by the purity of BG34, the molecular weight and chemical modification. In this study, we investigated the structural features of BG34 for macrophage recognition and internalization. We prepared homogeneous BG34s of 10 kDa (BG34-10), 200 kDa (BG34-200) and 500 kDa (BG34-500) with high purity, and then introduced green fluorescence FITC to the reducing ends (Re) or main chain (Mc). The results of size exclusion chromatography, 13C NMR, fluorescence microscopy, FACS analyses and MTS assay demonstrated that non-toxic BG34 of 10kDa (BG34-10) effectively trigger macrophage internalization. The internalization was adversely affected by modifying the main chain of BG34-10 but not the reducing end. Studies using blocking antibodies on several CD11b+ and CD11b− cells suggested that CD11b may play an important role in mediating macrophage internalization of BG34-10. Quantitative RT-PCR and intracellular cytokine stain revealed that macrophages generate increased level of CD11b and TNF-α in response to BG34-10. This study for the first time demonstrated the molecular size (10 kDa) and pattern of modification (reducing end modification) for BG34-10 to mediate macrophage internalization. Since BG34 is water soluble, biocompatible and biodegradable FDA-approved material, this mechanism of BG34-10 can be used to design drug delivery system targeting macrophages.
Introduction
It has been shown that β-glucans from yeast, fungi, grain (barley/oat) and seaweed adopt the Pathogen Associated Molecular Pattern (PAMP) so that they can trigger specific recognition and internalization by murine and human macrophages (Dergunova et al., 2009, Kawakami, 2009, Novak and Vetvicka, 2009, Rondanelli et al., 2009, Vetvicka and Vetvickova, 2009, Wu et al., 2010, Zhang et al., 2007). For this reason, β-glucans represent a very good candidate for the development of novel biomaterial targeting macrophages. Yeast glucan particles-mediated rapid internalization by macrophages through dectin-1 receptor are being investigated and the mechanism is applied to develop novel biomedical material targeting macrophages (Wu et al., 2010, Underhill, 2003). However, yeast glucans contain impurities of mannans, mannoproteins and chitin, and associate with water insoluble particles (Wu et al., 2010, Underhill, 2003). This has limited their development into well-characterized biomaterial with consistent biologic activity.
The β-(1 → 3)-(1 → 4)-glucans (BG34) from barley and oat are non-toxic, non-immunogenic FDA approved biomaterials with excellent water solubility, biocompatibility and biodegradability (Kawakami, 2009, Rondanelli et al., 2009, Xia, 1999, Tada et al., 2009a). It has been shown that BG34 can mediate receptor induced recognition and internalization by murine and human primary macrophages (Rondanelli et al., 2009, Xia, 1999, Tada et al., 2009a, Oliva et al., 2008, Brown et al., 2002, Brown, 2005, Tsoni and Brown, 2008), however, BG34s of different molecular weight (Mw), modification and various impurities appear to interact with macrophage differently (Mueller et al., 2000). The β-glucans of over 200 kDa may lead to the cross-linking with the cell membrane of macrophages, which results in a reduced uptake of BG34 by macrophages (Mueller et al., 2000). The hydroxyl groups in the main chain and aldehyde groups at the reducing end can provide substantial flexibility for modifying BG34 with functional groups, however, the modification may alter macrophage recognition and internalization as that BG34 functions as a PAMP molecule (Kawakami, 2009, Novak and Vetvicka, 2009, Wu et al., 2010, Zhang et al., 2007). For instances, studies by Xia (1999) suggested that a crude cell wall extract (contain 60–70% BG34 and insoluble cellulose) mediate CR3 (CD11b/CD18)-induced internalization by murine macrophages, while studies by Tada et al. (2009a) suggested that a purified glucan containing ∼75% BG34 mediated interaction with macrophages mainly through dectin-1 receptor.
Thus, in order to develop BG34-based material targeting macrophages, it is necessary to investigate the structural features of BG34s for effective macrophage internalization. In this study, we prepared homogeneous BG34 fractions of identical chemical structure, different Mw and modification patterns. Thioglycollate-elicited adherent mouse peritoneal cells were used as primary macrophages to examine macrophage internalization of soluble BG34s of various sizes and modifications. Our results suggested for the first time that BG34 of 10 kDa (BG34-10) effectively mediate macrophage internalization, and that the modification of the reducing end of BG34-10 does not interfere with such internalization. This has led to one of our ongoing studies in the development of cationic and pH-sensitive BG34-10 that can complex negatively charged therapeutic DNA/RNA into nanoparticles for gene delivery. Our preliminary studies have shown that the reducing end modification of BG34-10 using amines and peptides did not interference macrophage internalization.
Preliminary studies on the interaction of BG34-10 with myeloid derived suppressive cells (MDSCs) were conducted in this study. This is because high level of CD11b is expressed by MDSC (Kataru et al., 2009) and CD11b appears to play an important role in mediating macrophage uptake of BG34-10. More importantly, recent studies in animal models and humans have demonstrated that MDSCs are central regulator of tumor-induced immune suppression (Marigo et al., 2008). In healthy individual, MDSCs are few because immature myeloid cells (IMCs) generated in bone marrow quickly differentiate into mature granulocytes, macrophages and dendritic cells. By contrast, in tumor-bearing individual, tumor-secreted cytokines and/or chemokines can block the maturation of IMCs and promote the generation of MDSCs which can progressively accumulate in peripheral blood, lymphoid organs and migrate to tumor sites (Ugel et al., 2009, Ko, 2009). In addition to support angiogenesis and stromal remolding needed for tumor growth, MDSCs have a remarkable ability to suppress T cell-mediated anti-tumor responses (Gabrilovich and Nagaraj, 2009). As a result, malignant cancer cells survive, expand, subvert adaptive immunity, and reduce responses to therapy (Ugel et al., 2009, Ko, 2009). Recent clinical data has shown that an effective anti-tumor therapy must include a mechanism of reducing or eliminating MDSCs (Ugel et al., 2009, Ko, 2009). Therefore, our investigation on the interaction of BG34-10 with CD11b+ MDSCs may results in novel strategy targeting and eliminating MDSCs.
Macrophages and MDSCs have been implicated in the onset and/or progression of various diseases including cancer, autoimmune and inflammatory diseases (Siveen and Kuttan, 2009, Kinne et al., 2000, Burke et al., 2002, Gordon, 2007, Coffelt et al., 2009, Olefsky and Glass, 2010, Wilson, 2010), thus, the development of BG34-10 as novel biomaterial targeting macrophages will have significant implications for many disease conditions.
Section snippets
Materials
NHS-FITC and carbonyldiimidazole were purchased from Sigma. Anti-FITC antibody conjugated magnetic beads and strepavidin-conjugated magnetic beads were purchased from Thermo Pierce (Pierce, Rockford, USA). Anti-CD11b antibody pre-coated magnetic MicroBeads, MACS® LS Column and MACS Separator were purchased from Miltenyl Biotec (Miltenyl Biotec, Bergisch Gladbach, Germany). PMJ2R cell line was obtained from American Type Tissue Collection (ATCC, Rockville, MD, USA). Mw cut-off spin column (Mw
BG34s of 10, 200 and 500 kDa are highly purified and homogeneous glucan samples
In order to study the structural features of BG34s for macrophage recognition and internalization, we prepared highly purified and homogeneous BG34 samples of 10 kDa (BG34-10), 200 kDa (BG34-200) and 500 kDa (BG34-500) (Zhang et al., 2011). These BG34s were characterized by 13C NMR (Fig. 3A). The chemical shifts were assigned by comparing with published NMR data of oat β-glucan (Skendi, 2003). The result indicates that BG34-10, BG34-200, and BG34-500 adopt an identical chemical structure that is a
Discussion
The use of BG34 that contains impurities and contaminants has limitations in elucidating with certainty whether BG34 mediates specific recognition and internalization by macrophages. Studies by Tada used BG34 containing ∼25% α-glucans (Tada et al., 2009b). Studies by Xia used BG34 enriched from barley cell wall extract so that the BG34 contains insoluble cellulose (Xia et al., 1999a). Results of this current study demonstrated for the first time that the highly purified and homogeneous glucans
Acknowledgements
This study was supported by RGC project (2160365) from Hong Kong (M.Z.), NIH K23 CA109115-01A5 (J.K.), and University Hospitals Seidman Cancer Center Immunogene Therapy Fund (J.K.). We thank Case Comprehensive Cancer Center-Flow Core Lab for the kind assistance and support on FACS analysis.
References (50)
- S.B. Coffelt et al.Tumor-associated macrophages: effectors of angiogenesis and tumor progressionBiochim Biophys Acta(2009)
- M.A. Dergunova et al.Characterization of the novel chemically modified fungal polysaccharides as the macrophage stimulatorsInt Immunopharmacol(2009)
- J.-H. Kang et al.Liver-targeted siRNA delivery by polyethylenimine (PEI)-pullulan carrierBioorg Med Chem(2010)
- R.P. Kataru et al.Critical role of CD11b+ macrophages and VEGF in inflammatory lymphangiogenesis: antigen clearance, and inflammation resolutionBlood(2009)
- H.K. Le et al.Gemcitabine directly inhibits myeloid derived suppressor cells in BALB/c mice bearing 4T1 mammary carcinoma and augments expansion of T cells from tumor-bearing miceInt Immunopharmacol(2009)
- A. Mantovani et al.The chemokine system in diverse forms of macrophage activation and polarizationTrends Immunol(2004)
- K. Movahedi et al.Identification of discrete tumor-induced myeloid-derived suppressor cell subpopulations with distinct T cell-suppressive activityBlood(2008)
- A. Nueda et al.Hematopoietic cell-type-dependent regulation of leukocyte integrin functional activity: CD11b and CD11c expression inhibits LFA-1-dependent aggregation of differentiated U937 cellsCell Immunol(1995)
- K.S. Siveen et al.Role of macrophages in tumour progressionImmunol Lett(2009)
- A. SkendiStructure and rheological properties of water soluble β-glucans from oat cultivars of Avena sativa and Avena bysantinaJ Cereal Sci(2003)
References