IntroductionThe endogenous adenine nucleotides and adenosine are normally present at low concentrations in the extracellular milieu. However, metabolically stressful conditions, including inflammation and hypoxia characteristic of asthma, solid tumors, and other pathologic conditions, result in dramatic increases in extracellular concentrations of adenosine. [1][2][3] There are also mechanisms of nonlytic secretion of adenosine during hypoxic conditions.There is growing evidence that adenosine can actively modulate differentiation and function of myeloid cells. 4 Circulating cells of myeloid lineage, including monocytes and dendritic cell (DC) precursors, migrate to tissues where they differentiate into macrophages or DCs. DCs show impressive interaction with the adjacent microenvironment, 5,6 which regulates formation of DC subtypes and their functional properties, including expression of cytokines and growth factors. Because of rapid growth, solid tumors routinely experience severe hypoxia and necrosis, which causes adenine nucleotide degradation and adenosine release. Therefore, high levels of extracellular adenosine contribute to the local tumor microenvironment and may greatly influence differentiation of DCs from monocyte/macrophages and DC precursors migrating into tumor tissue. Adenosine acts through 4 subtypes of adenosine receptors, A 1 , A 2A , A 2B , and A 3 , which are members of the G-protein-coupled family of receptors. 7,8 A 2A adenosine receptors are generally anti-inflammatory, whereas A 2B and A 3 receptors are implicated in proinflammatory action of adenosine. Adenosine receptors are expressed abundantly on monocytes, and through these receptors adenosine exerts substantial modulatory effects on monocyte function and further differentiation. A 1 receptors were shown to stimulate formation of giant multinucleated cells from monocytes, whereas A 2 receptors inhibited this process. 9 A 2B receptors were implicated in mediating the inhibitory effect of adenosine on macrophage proliferation induced by M-CSF. 10 Exogenous adenosine can prevent monocytes from differentiating into macrophages, leading them to an intermediate differentiation stage between immature DCs and monocytes. 11 Cyclic nucleotides, including cAMP, which intracellular level increases in response to stimulation of adenosine A 2 receptors, regulate certain steps of monocyte differentiation and promote their differentiation toward a CD1a low CD14 ϩ/low CD209 ϩ intermediate cell but impair differentiation into functional DCs. 12 Up-regulation of DC-specific ICAM-3-grabbing nonintegrin (CD209) was not affected by cyclic nucleotides, 12 indicating that DC development was not blocked at the monocyte stage. The expression of all 4 adenosine receptor subtypes has been reported in human monocytes and myeloid DCs. 9,13-15 However, the effects of adenosine on differentiation of myeloid DCs from monocytes, macrophages, and hematopoietic progenitor cells (HPCs) and the roles of specific adenosine receptor subtypes involved in this process hav...

Vascular endothelial growth factor (VEGF), a major factor in tumor-host interactions, plays a critical role in the aberrant hematopoiesis observed in cancer-bearing hosts. To dissect the roles of VEGF receptor (VEGFR)-1 and VEGFR-2 in cancer-associated hematopoiesis in vivo, we selectively stimulated VEGFR-1 and VEGFR-2 by continuous infusion of receptor-specific ligands or selective blockade with VEGF receptor-specific antibodies in mice infused with recombinant VEGF at levels observed in tumor-bearing animals. We found that the effect of VEGF on the accumulation of Gr1+CD11b+ cells is mediated by VEGFR-2, but that the 2 receptors have opposite effects on lymphocyte development. Pathophysiologic levels of VEGF strongly inhibit T-cell development via VEGFR-2, whereas VEGFR-1 signaling decreases this inhibition. VEGFR-1, and not VEGFR-2, signaling is responsible for the observed increase of splenic B cells. Both receptors are capable of inhibiting dendritic cell function. These data suggest that most of observed aberrant hematopoiesis caused by excess tumor-derived VEGF is mediated by VEGFR-2, and VEGFR-1 alone has very limited independent effects but clearly both positively and negatively modulates the effects of VEGFR-2. Our findings suggest that selective blockade of VEGFR-2 rather than of both receptors may optimally overcome the adverse hematologic consequences of elevated VEGF levels found in malignancy.

Extracellular adenosine and purine nucleotides are elevated in many pathological situations associated with the expansion of CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs). Therefore, we tested whether adenosinergic pathways play a role in MDSC expansion and functions. We found that A2B adenosine receptors on hematopoietic cells play an important role in accumulation of intratumoral CD11b+Gr1high cells in a mouse Lewis lung carcinoma (LLC) model in vivo and demonstrated that these receptors promote preferential expansion of the granulocytic CD11b+Gr1high subset of MDSCs in vitro. Flow cytometry analysis of MDSCs generated from mouse hematopoietic progenitor cells revealed that the CD11b+Gr-1high subset had the highest levels of CD73 (ecto-5′-nucleotidase) expression (ΔMFI of 118.5±16.8), followed by CD11b+Gr-1int (ΔMFI of 57.9±6.8) and CD11b+Gr-1−/low (ΔMFI of 12.4±1.0). Even lower levels of CD73 expression were found on LLC tumor cells (ΔMFI of 3.2±0.2). The high levels of CD73 expression in granulocytic CD11b+Gr-1high cells correlated with high levels of ecto-5′-nucletidase enzymatic activity. We further demonstrated that the ability of granulocytic MDSCs to suppress CD3/CD28-induced T cell proliferation is significantly facilitated in the presence of the ecto-5′-nucletidase substrate 5′-AMP. We propose that generation of adenosine by CD73 expressed at high levels on granulocytic MDSCs may promote their expansion and facilitate their immunosuppressive activity.

Impaired Ag-presenting function in dendritic cells (DCs) due to abnormal differentiation is an important mechanism of tumor escape from immune control. A major role for vascular endothelial growth factor (VEGF) and its receptors, VEGFR1/Flt-1 and VEGFR2/KDR/Flk-1, has been documented in hemopoietic development. To study the roles of each of these receptors in DC differentiation, we used an in vitro system of myeloid DC differentiation from murine embryonic stem cells. Exposure of wild-type, VEGFR1−/−, or VEGFR2−/− embryonic stem cells to exogenous VEGF or the VEGFR1-specific ligand, placental growth factor, revealed distinct roles of VEGF receptors. VEGFR1 is the primary mediator of the VEGF inhibition of DC maturation, whereas VEGFR2 tyrosine kinase signaling is essential for early hemopoietic differentiation, but only marginally affects final DC maturation. SU5416, a VEGF receptor tyrosine kinase inhibitor, only partially rescued the mature DC phenotype in the presence of VEGF, suggesting the involvement of both tyrosine kinase-dependent and independent inhibitory mechanisms. VEGFR1 signaling was sufficient for blocking NF-κB activation in bone marrow hemopoietic progenitor cells. VEGF and placental growth factor affect the early stages of myeloid/DC differentiation. The data suggest that therapeutic strategies attempting to reverse the immunosuppressive effects of VEGF in cancer patients might be more effective if they specifically targeted VEGFR1.

Recent studies suggest that tumor-infiltrating immune cells can benefit the tumor by producing factors that promote angiogenesis and suppress immunity. Because the tumor microenvironment is characterized by high adenosine levels, we hypothesized that the low-affinity A(2B) adenosine receptor located on host immune cells may participate in these effects. In the current study, we tested this hypothesis in a Lewis lung carcinoma isograft model using A(2B) receptor knockout (A(2B)KO) mice. These mice exhibited significantly attenuated tumor growth and longer survival times after inoculation with Lewis lung carcinoma compared to wild type (WT) controls. Lewis lung carcinoma tumors in A(2B)KO mice contained significantly lower levels of vascular endothelial growth factor (VEGF) compared to tumors growing in WT animals. This difference was due to VEGF production by host cells, which comprised 30 +/- 2% of total tumor cell population. Stimulation of adenosine receptors on WT tumor-infiltrating CD45+ immune cells increased VEGF production fivefold, an effect not seen in tumor-associated CD45+ immune cells lacking A(2B) receptors. In contrast, we found no significant difference in VEGF production between CD45- tumor cells isolated from WT and A(2B)KO mice. Thus, our data suggest that tumor cells promote their growth by exploiting A(2B) adenosine receptor-dependent regulation of VEGF in host immune cells.