Inflammation and angiogenesis are pivotal processes in the progression of many diseases, including malignancies. A hypoxic microenvironment often results in a milieu of proinflammatory and proangiogenic cytokines produced by infiltrating cells. We assessed the role of macrophage-derived hypoxia-associated cytokines in promoting inflammation and angiogenesis. Supernatants of macrophages, stimulated under hypoxia with or without an inflammatory stimulus (LPS), promoted angiogenesis when incorporated into Matrigel plugs. However, neutralization of IL-1 in the supernatants, particularly IL-1β, completely abrogated cell infiltration and angiogenesis in Matrigel plugs and reduced vascular endothelial growth factor (VEGF) levels by 85%. Similarly, supernatants from macrophages of IL-1β knockout mice did not induce inflammatory or angiogenic responses. The importance of IL-1 signaling in the host was demonstrated by the dramatic reduction of inflammatory and angiogenic responses in Matrigel plugs that contained macrophage supernatants from control mice which had been implanted in IL-1 receptor type I knockout mice. Myeloid cells infiltrating into Matrigel plugs were of bone marrow origin and represented the major source of IL-1 and other cytokines/chemokines in the plugs. Cells of endothelial lineage were the main source of VEGF and were recruited mainly from neighboring tissues, rather than from the bone marrow. Using the aortic ring sprouting assay, it was shown that in this experimental system, IL-1 does not directly activate endothelial cell migration, proliferation and organization into blood vessel-like structures, but rather activates infiltrating cells to produce endothelial cell activating factors, such as VEGF. Thus, targeting IL-1β has the potential to inhibit angiogenesis in pathological situations and may be of considerable clinical value.

Regulating immunity is a leading target for cancer therapy. Here, we show that the anti-tumor immune response can be modulated by the brain’s reward system, a key circuitry in emotional processes. Activation of the reward system in tumor-bearing mice (Lewis lung carcinoma (LLC) and B16 melanoma) using chemogenetics (DREADDs), resulted in reduced tumor weight. This effect was mediated via the sympathetic nervous system (SNS), manifested by an attenuated noradrenergic input to a major immunological site, the bone marrow. Myeloid derived suppressor cells (MDSCs), which develop in the bone marrow, became less immunosuppressive following reward system activation. By depleting or adoptively transferring the MDSCs, we demonstrated that these cells are both necessary and sufficient to mediate reward system effects on tumor growth. Given the central role of the reward system in positive emotions, these findings introduce a physiological mechanism whereby the patient’s psychological state can impact anti-tumor immunity and cancer progression.

Tumor cell-macrophage interactions change as the tumor progresses, and the generation of nitric oxide (NO) by the inducible nitric oxide synthase (iNOS) plays a major role in this interplay. In early stages, macrophages employ their killing mechanisms, particularly the generation of high concentrations of NO and its derivative reactive nitrogen species (RNS) to initiate tumor cell apoptosis and destroy emerging transformed cells. If the tumor escapes the immune system and grows, macrophages that infiltrate it are reprogramed in situ by the tumor microenvironment. Low oxygen tensions (hypoxia) and immunosuppressive cytokines inhibit iNOS activity and lead to production of low amounts of NO/RNS, which are pro-angiogenic and support tumor growth and metastasis by inducing growth factors (e.g., VEGF) and matrix metalloproteinases (MMPs). We review here the different roles of NO/RNS in tumor progression and inhibition, and the mechanisms that regulate iNOS expression and NO production, highlighting the role of different subtypes of macrophages and the microenvironment. We finally claim that some tumor cells may become resistant to macrophage-induced death by increasing their expression of microRNA-146a (miR-146a), which leads to inhibition of iNOS translation. This implies that some cooperation between tumor cells and macrophages is required to induce tumor cell death, and that tumor cells may control their fate. Thus, in order to induce susceptibility of tumors cells to macrophage-induced death, we suggest a new therapeutic approach that couples manipulation of miR-146a levels in tumors with macrophage therapy, which relies on ex vivo stimulation of macrophages and their re-introduction to tumors.

The inflammatory response is marked by the release of several cytokines with multiple roles in regulating leukocyte activities, including the secretion of matrix metalloproteinases (MMPs). Although the effects of individual cytokines on monocyte MMP expression have been studied extensively, few studies have examined the influence of combinations of cytokines, which are likely present at inflammatory sites. Herein, we report our investigation of the combinatorial effects of tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β on MMP-9 synthesis. We found that TGF-β suppressed TNF-α-induced MMP-9 secretion by MonoMac-6 monocytic cells in a dose-dependent manner, with a maximal effect of TGF-β observed at 1 ng/ml. Such suppression was likely regulated at the pretranslational level, because steady-state mRNA levels of TNF-α-induced MMP-9 were reduced by TGF-β, and pulse-chase radiolabeling also showed a decrease in new MMP-9 protein synthesis. The suppressive effects of TGF-β were time dependent, because short exposures to TNF-α before TGF-β or simultaneous exposure to both cytokines efficiently reduced MMP-9 secretion. Expression of the tissue inhibitor of metalloproteinases (TIMP)-1 and TNF-α receptors was unaffected by either cytokine individually or in combination. Affinity binding with radiolabeled TGF-β demonstrated that levels of TGF-β receptors were not increased after preincubation with TGF-β. Suppression of TNFα-induced MMP-9 secretion by TGF-β correlated with a reduction in prostaglandin E2 (PGE2) secretion. Furthermore, the effect of TGF-β or indomethacin on blockage of TNF-α-stimulated MMP-9 production was reversed by the addition of either exogenous PGE2 or the cyclic AMP (cAMP) analogue Bt2cAMP. Thus, we concluded that TGF-β acts as a potent suppressor of TNF-α-induced monocyte MMP-9 synthesis via a PGE2- and cAMP-dependent mechanism. These results suggest that various combinations of cytokines that are present at inflammatory sites, as well as their balance during different stages of inflammation, may provide the signals necessary for directing MMP-mediated leukocyte activities.

Cellular hypoxia, characterizing tumors, ischemia, and inflammation induce recruitment of monocytes/macrophages, immobilize them at the hypoxic site, and alter their function. To migrate across the extracellular matrix and as part of their inflammatory functions, monocytes and macrophages secrete proteases, including matrix metalloproteinase-9 (MMP-9), whose expression is induced by proinflammatory cytokines [e.g., tumor necrosis factor α (TNF-α)]. We show that hypoxia (<0.3% O₂ for 48 h) reduced the output of TNF-α-induced proMMP-9 by threefold (P<0.01) in the U937 monocytic cell line and in primary human monocytes. TNF-α induced MMP-9 transcription by threefold, but no significant difference was observed in MMP-9 mRNA steady-state between normoxia and hypoxia, which inhibited the trafficking of proMMP-9 via secretory vesicles and increased the intracellular accumulation of proMMP-9 in the cells by 47% and 62% compared with normoxia (P<0.05), as evaluated by zymography of cellular extracts and confocal microscopy, respectively. Secretion of proMMP-9 was reduced by the addition of cytochalazin B or nocodazole, which inhibits the polymerization of actin and tubulin fibers, or by the addition of the Rho kinase inhibitor Y27632, suggesting the involvement of the cytoskeleton and the Rho GTPases in the process of enzyme secretion. Furthermore, attachment of proMMP-9 to the cell membrane increased after hypoxia via its interactions with surface molecules such as CD44. In addition, the reduced migration of monocytes in hypoxia was shown to be mediated, at least partially, by secreted MMP-9. Thus, hypoxia post-translationally reduced the secreted amounts of proMMP-9 by using two mutually nonexclusive mechanisms: mostly, inhibition of cellular trafficking and to a lesser extent, attachment to the membrane.

Tumor necrosis factor α (TNF-α) is a proinflammatory cytokine implicated in the stimulation of matrix metalloproteinase (MMP) production by several cell types. Our previous studies demonstrated that TNF-α avidly binds fibronectin (FN) and laminin, major adhesive glycoproteins of extracellular matrix (ECM) and basement membranes. These findings suggested that TNF-α complexing to insoluble ECM components may serve to concentrate its activities to distinct inflamed sites. Herein, we explored the bioactivity and possible function of ECM-bound TNF-α by examining its effects on MMP-9 secretion by monocytes. Immunofluorescent staining indicated that LPS-activated monocytes deposited newly synthesized TNF-α into ECM-FN. FN-bound TNF-α (FN/TNF-α) significantly up-regulated MMP-9 expression and secretion by the human monocytic cell line MonoMac-6 and peripheral blood monocytes. Such secretion could be inhibited by antibodies that block TNF-α activity and binding to its receptors TNF RI (p55) and TNF RII (p75). Chemotaxis through ECM gels in the presence of soluble or bound TNF-α was inhibited by a hydroxamic acid inhibitor of MMPs (GM6001). It is interesting that, although the adhesion of MonoMac-6 cells to FN/TNF-α required functional activated β1 integrins, FN/TNF-α-induced MMP-9 secretion was independent of binding to β1 integrins, since MMP-9 secretion was unaffected by: (1) neutralizing mAb to α4, α5, and β1 subunits, which blocked cell adhesion; (2) a mAb that stimulated β1 integrin-mediated adhesion; and (3) binding TNF-α to the 30-kDa amino-terminal fragment of FN, which lacks the major cell adhesive binding sites. Thus, in addition to their cell-adhesive roles, ECM glycoproteins, such as FN, may play a pivotal role in presenting proinflammatory cytokines to leukocytes within the actual inflamed tissue, thereby affecting their capacities to secrete ECM-degrading enzymes. These TNF-α-ECM interactions may serve to limit the cytokine’s availability and bioactivity to target areas of inflammation.