The role of IL-1ß in the early tumor cell-induced angiogenic response.

In this study, we assessed the involvement of IL-1ß in early angiogenic responses induced by malignant cells using Matrigel plugs supplemented with B16 melanoma cells. We found that during the angiogenic response, IL-1ß and vascular endothelial growth factor (VEGF) interact in a newly described autoinduction circuit, in which each of these cytokines induces the other. The IL-1ß and VEGF circuit acts through interactions between bone marrow-derived VEGF receptor 1(+)/IL-1R1(+) immature myeloid cells and tissue endothelial cells. Myeloid cells produce IL-1ß and additional proinflammatory cytokines, which subsequently activate endothelial cells to produce VEGF and other proangiogenic factors and provide the inflammatory microenvironment for angiogenesis and tumor progression. These mechanisms were also observed in a nontumor early angiogenic response elicited in Matrigel plugs by either rIL-1ß or recombinant VEGF. We have shown that IL-1ß inhibition stably reduces tumor growth by limiting inflammation and inducing the maturation of immature myeloid cells into M1 macrophages. In sharp contrast, only transient inhibition of tumor growth was observed after VEGF neutralization, followed by tumor recurrence mediated by rebound angiogenesis. This occurs via the reprogramming of VEGF receptor 1(+)/IL-1R1(+) cells to express hypoxia inducible factor-1α, VEGF, and other angiogenic factors, thereby directly supporting proliferation of endothelial cells and blood vessel formation in a paracrine manner. We suggest using IL-1ß inhibition as an effective antitumor therapy and are currently optimizing the conditions for its application in the clinic.

IL-1α and IL-1ß recruit different myeloid cells and promote different stages of sterile inflammation.

The immune system has evolved to protect the host from invading pathogens and to maintain tissue homeostasis. Although the inflammatory process involving pathogens is well documented, the intrinsic compounds that initiate sterile inflammation and how its progression is mediated are still not clear. Because tissue injury is usually associated with ischemia and the accompanied hypoxia, the microenvironment of various pathologies involves anaerobic metabolites and products of necrotic cells. In the current study, we assessed in a comparative manner the role of IL-1α and IL-1ß in the initiation and propagation of sterile inflammation induced by products of hypoxic cells. We found that following hypoxia, the precursor form of IL-1α, and not IL-1ß, is upregulated and subsequently released from dying cells. Using an inflammation-monitoring system consisting of Matrigel mixed with supernatants of hypoxic cells, we noted accumulation of IL-1α in the initial phase, which correlated with the infiltration of neutrophils, and the expression of IL-1ß correlated with later migration of macrophages. In addition, we were able to show that IL-1 molecules from cells transfected with either precursor IL-1α or mature IL-1ß can recruit neutrophils or macrophages, respectively. Taken together, these data suggest that IL-1α, released from dying cells, initiates sterile inflammation by inducing recruitment of neutrophils, whereas IL-1ß promotes the recruitment and retention of macrophages. Overall, our data provide new insight into the biology of IL-1 molecules as well as on the regulation of sterile inflammation.

Differential release of chromatin-bound IL-1alpha discriminates between necrotic and apoptotic cell death by the ability to induce sterile inflammation.

IL-1alpha, like IL-1beta, possesses multiple inflammatory and immune properties. However, unlike IL-1beta, the cytokine is present intracellularly in healthy tissues and is not actively secreted. Rather, IL-1alpha translocates to the nucleus and participates in transcription. Here we show that intracellular IL-1alpha is a chromatin-associated cytokine and highly dynamic in the nucleus of living cells. During apoptosis, IL-1alpha concentrates in dense nuclear foci, which markedly reduces its mobile nature. In apoptotic cells, IL-1alpha is retained within the chromatin fraction and is not released along with the cytoplasmic contents. To simulate the in vivo inflammatory response to cells undergoing different mechanisms of death, lysates of cells were embedded in Matrigel plugs and implanted into mice. Lysates from cells undergoing necrosis recruited cells of the myeloid lineage into the Matrigel, whereas lysates of necrotic cells lacking IL-1alpha failed to recruit an infiltrate. In contrast, lysates of cells undergoing apoptotic death were inactive. Cells infiltrating the Matrigel were due to low concentrations (20-50 pg) of the IL-1alpha precursor containing the receptor interacting C-terminal, whereas the N-terminal propiece containing the nuclear localization site failed to do so. When normal keratinocytes were subjected to hypoxia, the constitutive IL-1alpha precursor was released into the supernatant. Thus, after an ischemic event, the IL-1alpha precursor is released by hypoxic cells and incites an inflammatory response by recruiting myeloid cells into the area. Tissues surrounding the necrotic site also sustain damage from the myeloid cells. Nuclear trafficking and differential release during necrosis vs. apoptosis demonstrate that inflammation by IL-1alpha is tightly controlled.

The role of macrophage-derived IL-1 in induction and maintenance of angiogenesis.

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-1beta, 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-1beta 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-1beta has the potential to inhibit angiogenesis in pathological situations and may be of considerable clinical value.

Host-derived interleukin-1alpha is important in determining the immunogenicity of 3-methylcholantrene tumor cells.

Using IL-1/IL-1Ra knockout BALB/c mice, we showed that 3-methylcholatrene (3-MCA)-induced carcinogenesis is dependent on IL-1beta-induced inflammatory responses. Patterns of local inflammation and tumorigenicity were similar in wild-type (WT) and IL-1alpha(-/-) mice, while in IL-1beta(-/-) mice, tumorigenicity was attenuated and in IL-1Ra(-/-) mice accentuated. 3-MCA-induced fibrosarcoma cell lines from WT mice developed into progressive tumors in WT mice, while surprisingly, lines from IL-1alpha(-/-) mice formed tumors only in immunocompromized mice. 3-MCA-induced fibrosarcoma cell lines from IL-1alpha(-/-) mice, compared with lines from WT mice, manifested higher expression levels of "global" surface molecules related to Ag presentation and interactions with immune surveillance cells (MHC class I, B7.1, B7.2, L-selectin, and NKG2D ligands) and were eradicated mainly by CD4(+)- and CD8(+)-dependent T cell responses. Concomitantly, at the injection site of 3-MCA-induced fibrosarcoma cells derived from IL-1alpha(-/-) mice, a leukocyte infiltrate, subsequently replaced by a scar-like tissue, was observed. Immune aberrations in NK cell maturation, antitumor specific immunity and killing capacity of effector cells were observed in IL-1alpha(-/-) mice, in contrast to WT mice. Thus, we demonstrate in this study the significance of host-derived IL-1alpha in cancer immunoediting, by affecting innate and specific immunosurveillance mechanisms. Overall, the results presented in this study, together with our previous studies, attest to differential involvement of IL-1alpha and IL-1beta in tumorigenesis; host-derived IL-1beta mainly controls inflammation, while concomitantly, IL-1alpha controls immunosurveillance of the arising malignant cells. Elucidation of the involvement of the IL-1 molecules in the malignant process will hopefully lead to the development of novel approaches for chemoprevention and immunotherapy.

The involvement of IL-1 in tumorigenesis, tumor invasiveness, metastasis and tumor-host interactions.

Interleukin-1 (IL-1) includes a family of closely related genes; the two major agonistic proteins, IL-1alpha and IL-1beta, are pleiotropic and affect mainly inflammation, immunity and hemopoiesis. The IL-1Ra antagonist is a physiological inhibitor of pre-formed IL-1. Recombinant IL-1alpha and IL-1beta bind to the same receptors and induce the same biological functions. As such, the IL-1 molecules have been considered identical in normal homeostasis and in disease. However, the IL-1 molecules differ in their compartmentalization within the producing cell or the microenvironment. Thus, IL-1beta is solely active in its secreted form, whereas IL-1alpha is mainly active in cell-associated forms (intracellular precursor and membrane-bound IL-1alpha) and only rarely as a secreted cytokine, as it is secreted only in a limited manner. IL-1 is abundant at tumor sites, where it may affect the process of carcinogenesis, tumor growth and invasiveness and also the patterns of tumor-host interactions. Here, we review the effects of microenvironment- and tumor cell-derived IL-1 on malignant processes in experimental tumor models and in cancer patients. We propose that membrane-associated IL-1alpha expressed on malignant cells stimulates anti-tumor immunity, while secretable IL-1beta, derived from the microenvironment or the malignant cells, activates inflammation that promotes invasiveness and also induces tumor-mediated suppression. Inhibition of the function of IL-1 by the IL-1Ra, reduces tumor invasiveness and alleviates tumor-mediated suppression, pointing to its feasibility in cancer therapy. Differential manipulation of IL-1alpha and IL-1beta in malignant cells or in the tumor's microenvironment can open new avenues for using IL-1 in cancer therapy.