Elena Voronov

IL-1 is required for tumor invasiveness and angiogenesis

Here, we describe that microenvironmental IL-1β and, to a lesser extent, IL-1α are required for in vivo angiogenesis and invasiveness of different tumor cells. In IL-1β knockout (KO) mice, local tumor or lung metastases of B16 melanoma cells were not observed compared with WT mice. Angiogenesis was assessed by the recruitment of blood vessel networks into Matrigel plugs containing B16 melanoma cells; vascularization of the plugs was present in WT mice, but was absent in IL-1β KO mice. The addition of exogenous IL-1 into B16-containing Matrigel plugs in IL-1β KO mice partially restored the angiogenic response. Moreover, the incorporation of IL-1 receptor antagonist to B16-containing plugs in WT mice inhibited the ingrowth of blood vessel networks into Matrigel plugs. In IL-1α KO mice, local tumor development and induction of an angiogenic response in Matrigel plugs was less pronounced than in WT mice, but significantly higher than in IL-1β KO mice. These effects of host-derived IL-1α and IL-1β were not restricted to the melanoma model, but were also observed in DA/3 mammary and prostate cancer cell models. In addition to the in vivo findings, IL-1 contributed to the production of vascular endothelial cell growth factor and tumor necrosis factor in cocultures of peritoneal macrophages and tumor cells. Host-derived IL-1 seems to control tumor angiogenesis and invasiveness. Furthermore, the anti-angiogenic effects of IL-1 receptor antagonist, shown here, suggest a possible therapeutic role in cancer, in addition to its current use in rheumatoid arthritis.

IL-1α and IL-1β 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-1β 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-1β, 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-1β 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-1β 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-1β 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-1α discriminates between necrotic and apoptotic cell death by the ability to induce sterile inflammation

IL-1α, like IL-1β, possesses multiple inflammatory and immune properties. However, unlike IL-1β, the cytokine is present intracellularly in healthy tissues and is not actively secreted. Rather, IL-1α translocates to the nucleus and participates in transcription. Here we show that intracellular IL-1α is a chromatin-associated cytokine and highly dynamic in the nucleus of living cells. During apoptosis, IL-1α concentrates in dense nuclear foci, which markedly reduces its mobile nature. In apoptotic cells, IL-1α 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-1α 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-1α 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-1α precursor was released into the supernatant. Thus, after an ischemic event, the IL-1α 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-1α is tightly controlled.

CD11b+/Gr-1+ Immature Myeloid Cells Mediate Suppression of T Cells in Mice Bearing Tumors of IL-1β-Secreting Cells

Tumor cells secreting IL-1β are invasive and metastatic, more than the parental line or control mock-transfected cells, and concomitantly induce in mice general immune suppression of T cell responses. Suppression strongly correlates with accumulation in the peripheral blood and spleen of CD11b+/Gr-1+ immature myeloid cells and hematological alterations, such as splenomegaly, leukocytosis, and anemia. Resection of large tumors of IL-1β-secreting cells restored immune reactivity and hematological alterations within 7–10 days. Treatment of tumor-bearing mice with the physiological inhibitor of IL-1, the IL-1R antagonist, reduced tumor growth and attenuated the hematological alterations. Depletion of CD11b+/Gr-1+ immature myeloid cells from splenocytes of tumor-bearing mice abrogated suppression. Despite tumor-mediated suppression, resection of large tumors of IL-1β-secreting cells, followed by a challenge with the wild-type parental cells, induced resistance in mice; protection was not observed in mice bearing tumors of mock-transfected fibrosarcoma cells. Altogether, we show in this study that tumor-derived IL-1β, in addition to its proinflammatory effects on tumor invasiveness, induces in the host hematological alterations and tumor-mediated suppression. Furthermore, the antitumor effectiveness of the IL-1R antagonist was also shown to encompass restoration of hematological alterations, in addition to its favorable effects on tumor invasiveness and angiogenesis that have previously been described by us.

Interleukin-1β–Driven Inflammation Promotes the Development and Invasiveness of Chemical Carcinogen–Induced Tumors

The role of microenvironment interleukin 1 (IL-1) on 3-methylcholanthrene (3-MCA)-induced carcinogenesis was assessed in IL-1-deficient mice, i.e., IL-1beta(-/-), IL-1alpha(-/-), IL-1alpha/beta(-/-) (double knockout), and mice deficient in the naturally occurring inhibitor of IL-1, the IL-1 receptor antagonist (IL-1Ra). Tumors developed in all wild-type (WT) mice, whereas in IL-1beta-deficient mice, tumors developed slower and only in some of the mice. In IL-1Ra-deficient mice, tumor development was the most rapid. Tumor incidence was similar in WT and IL-1alpha-deficient mice. Histologic analyses revealed fibrotic structures forming a capsule surrounding droplets of the carcinogen in olive oil, resembling foreign body-like granulomas, which appeared 10 days after injection of 3-MCA and persisted until the development of local tumors. A sparse leukocyte infiltrate was found at the site of carcinogen injection in IL-1beta-deficient mice, whereas in IL-1Ra-deficient mice, a dense neutrophilic infiltrate was observed. Treatment of IL-1Ra-deficient mice with recombinant IL-1Ra but not with an inhibitor of tumor necrosis factor abrogated the early leukocytic infiltrate. The late leukocyte infiltrate (day 70), which was dominated by macrophages, was also apparent in WT and IL-1alpha-deficient mice, but was nearly absent in IL-1beta-deficient mice. Fibrosarcoma cell lines, established from 3-MCA-induced tumors from IL-1Ra-deficient mice, were more aggressive and metastatic than lines from WT mice; cell lines from IL-1-deficient mice were the least invasive. These observations show the crucial role of microenvironment-derived IL-1beta, rather than IL-1alpha, in chemical carcinogenesis and in determining the invasive potential of malignant cells.

Interleukin-1—a major pleiotropic cytokine in tumor–host interactions

Interleukin-1 (IL-1) represents a family of two agonistic proteins, IL-1alpha and IL-1beta, that are pleiotropic and affect hemopoiesis, inflammation, and immunity. In the context of the producing cell, IL-1beta is solely active in its secreted form, whereas IL-1alpha is active as an intracellular precursor, as a membrane-associated cytokine and to a lesser extent as a secreted molecule. IL-1 is abundant at tumor sites, where it may not only affect the growth and invasiveness of malignant cells, but where it may also induce antitumor immunity. Here we review the effects of microenvironmental and tumor cell-associated IL-1 on malignant processes, in experimental tumor models and in cancer patients.

Is interleukin-1 a good or bad ‘guy’ in tumor immunobiology and immunotherapy?

Summary: The interleukin‐1 (IL‐1) family consists of two major agonistic proteins, IL‐1α and IL‐1β, which are pleiotropic and affect mainly inflammation, immunity, and hemopoiesis. The IL‐1 receptor antagonist (IL‐1Ra) is a physiological inhibitor of pre‐formed IL‐1. In their secreted form, IL‐1α and IL‐1β bind to the same receptors and induce the same biological functions. However, the IL‐1 molecules differ in their compartmentalization within the producing cell or the microenvironment. Thus, IL‐1β is solely active in its secreted form, whereas IL‐1α is mainly active in cell‐associated forms (intracellular precursor and membrane‐bound IL‐1) and only rarely as a secreted cytokine, mainly by macrophages/monocytes. IL‐1 is abundant at tumor sites, being produced by cellular elements of the tumor microenvironment or by the malignant cells, and it affects not only various phases of the malignant process, such as carcinogenesis, tumor growth, and invasiveness, but also patterns of interactions between malignant cells and the hosts immune system. Hence, the effects of the IL‐1 molecules on the malignant process are complex and are often of an opposing nature. Comparative studies on the differential roles of malignant cell‐ or host‐derived IL‐1α and IL‐1β in different stages of the malignant process can subsequently open new avenues for manipulation of IL‐1 expression and function in cancer immunotherapy.

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-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.

Differential effects of IL-1 alpha and IL-1 beta on tumorigenicity patterns and invasiveness

In this study, we show that distinct compartmentalization patterns of the IL-1 molecules (IL-1α and IL-1β), in the milieu of tumor cells that produce them, differentially affect the malignant process. Active forms of IL-1, namely precursor IL-1α (pIL-1α), mature IL-1β (mIL-1β), and mIL-1β fused to a signal sequence (ssIL-1β), were transfected into an established fibrosarcoma cell line, and tumorigenicity and antitumor immunity were assessed. Cell lines transfected with pIL-1α, which expresses IL-1α on the membrane, fail to develop local tumors and activate antitumor effector mechanisms, such as CTLs, NK cells, and high levels of IFN-γ production. Cells transfected with secretable IL-1β (mIL-1β and ssIL-1β) were more aggressive than wild-type and mock-transfected tumor cells; ssIL-1β transfectants even exhibited metastatic tumors in the lungs of mice after i.v. inoculation (experimental metastasis). In IL-1β tumors, increased vascularity patterns were observed. No detectable antitumor effector mechanisms were observed in spleens of mice injected with IL-1β transfectants, mock-transfected or wild-type fibrosarcoma cells. Moreover, in spleens of mice injected with IL-1β transfectants, suppression of polyclonal mitogenic responses (proliferation, IFN-γ and IL-2 production) to Con A was observed, suggesting the development of general anergy. Histologically, infiltrating mononuclear cells penetrating the tumor were seen at pIL-1α tumor sites, whereas in mIL-1β and ssIL-1β tumor sites such infiltrating cells do not penetrate inside the tumor. This is, to our knowledge, the first report on differential, nonredundant, in vivo effects of IL-1α and IL-1β in malignant processes; IL-1α reduces tumorigenicity by inducing antitumor immunity, whereas IL-1β promotes invasiveness, including tumor angiogenesis, and also induces immune suppression in the host.

Monocytes-macrophages that express α-smooth muscle actin preserve primitive hematopoietic cells in the bone marrow

Hematopoietic stem and progenitor cells (HSPCs) are regulated by various bone marrow stromal cell types. Here we identified rare activated bone marrow monocytes and macrophages with high expression of α-smooth muscle actin (α-SMA) and the cyclooxygenase COX-2 that were adjacent to primitive HSPCs. These myeloid cells resisted radiation-induced cell death and further upregulated COX-2 expression under stress conditions. COX-2-derived prostaglandin E2 (PGE2) prevented HSPC exhaustion by limiting the production of reactive oxygen species (ROS) via inhibition of the kinase Akt and higher stromal-cell expression of the chemokine CXCL12, which is essential for stem-cell quiescence. Our study identifies a previously unknown subset of α-SMA+ activated monocytes and macrophages that maintain HSPCs and protect them from exhaustion during alarm situations.