IL-1β Deficient Mice Research Articles

IL1β Promotes Immune Suppression in the Tumor Microenvironment Independent of the Inflammasome and Gasdermin D.

IL1β is a central mediator of inflammation. Secretion of IL1β typically requires proteolytic maturation by the inflammasome and formation of membrane pores by gasdermin D (GSDMD). Emerging evidence suggests an important role for IL1β in promoting cancer progression in patients, but the underlying mechanisms are ill-defined. Here, we have shown a key role for IL1β in driving tumor progression in two distinct mouse tumor models. Notably, activation of the inflammasome, caspase-8, as well as the pore-forming proteins GSDMD and mixed lineage kinase domain-like protein in the host were dispensable for the release of intratumoral bioactive IL1β. Inflammasome-independent IL1β release promoted systemic neutrophil expansion and fostered accumulation of T-cell-suppressive neutrophils in the tumor. Moreover, IL1β was essential for neutrophil infiltration triggered by antiangiogenic therapy, thereby contributing to treatment-induced immunosuppression. Deletion of IL1β allowed intratumoral accumulation of CD8+ effector T cells that subsequently activated tumor-associated macrophages. Depletion of either CD8+ T cells or macrophages abolished tumor growth inhibition in IL1β-deficient mice, demonstrating a crucial role for CD8+ T-cell-macrophage cross-talk in the antitumor immune response. Overall, these results support a tumor-promoting role for IL1β through establishing an immunosuppressive microenvironment and show that inflammasome activation is not essential for release of this cytokine in tumors.

AIM2 inflammasome-derived IL-1\u03b2 induces postoperative ileus in mice

Postoperative ileus (POI) is an intestinal dysmotility frequently occurring after abdominal surgery. An orchestrated neuroimmune response within the muscularis externa (ME) involves activation of resident macrophages, enteric glia and infiltration of blood-derived leukocytes. Interleukin-1 receptor type-I (IL1R1) signalling on enteric glia has been shown to be involved in POI development. Herein we investigated the distinct role of the IL1R1 ligands interleukin (IL) -1α and IL-1β and focused on the mechanism of IL-1β production. IL-1α and IL-1β deficient mice were protected from POI. Bone-marrow transplantation studies indicated that IL-1α originated from radio-resistant cells while IL-1β was released from the radio-sensitive infiltrating leukocytes. Mouse strains deficient in inflammasome formation identified the absent in melanoma 2 (AIM2) inflammasome to be crucial for IL-1β production in POI. Mechanistically, antibiotic-treated mice revealed a prominent role of the microbiome in IL-1β production. Our study provides new insights into distinct roles of IL-1α and IL-1β signalling during POI. While IL-1α release is most likely an immediate passive response to the surgical trauma, IL-1β production depends on AIM2 inflammasome formation and the microbiome. Selective interaction in this pathway might be a promising target to prevent POI in surgical patients.

Saturated Fatty Acids Undergo Intracellular Crystallization and Activate the NLRP3 Inflammasome in Macrophages

Inflammation provoked by the imbalance of fatty acid composition, such as excess saturated fatty acids (SFAs), is implicated in the development of metabolic diseases. Recent investigations suggest the possible role of the NLRP3 (nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3) inflammasome, which regulates IL-1β (interleukin 1β) release and leads to inflammation, in this process. Therefore, we investigated the underlying mechanism by which SFAs trigger NLRP3 inflammasome activation. The treatment with SFAs, such as palmitic acid and stearic acid, promoted IL-1β release in murine primary macrophages while treatment with oleic acid inhibited SFA-induced IL-1β release in a dose-dependent manner. Analyses using polarized light microscopy revealed that intracellular crystallization was provoked in SFA-treated macrophages. As well as IL-1β release, the intracellular crystallization and lysosomal dysfunction were inhibited in the presence of oleic acid. These results suggest that SFAs activate NLRP3 inflammasome through intracellular crystallization. Indeed, SFA-derived crystals activated NLRP3 inflammasome and subsequent IL-1β release via lysosomal dysfunction. Excess SFAs also induced crystallization and IL-1β release in vivo. Furthermore, SFA-derived crystals provoked acute inflammation, which was impaired in IL-1β-deficient mice. These findings demonstrate that excess SFAs cause intracellular crystallization and subsequent lysosomal dysfunction, leading to the activation of the NLRP3 inflammasome, and provide novel insights into the pathogenesis of metabolic diseases.

Interleukin-1α and Interleukin-1β play a central role in the pathogenesis of fulminant hepatic failure in mice.

Background and aimsFulminant hepatitis failure (FHF) is marked by the sudden loss of hepatic function, with a severe life-threatening course in persons with no prior history of liver disease. Interleukin (IL)-1α and IL-1β are key inflammatory cytokines but little is known about their role in the development of FHF. The aim of this study was to assess the involvement of IL-1α and IL-1β in the progression of LPS/GalN-induced FHF.MethodsWT, IL-1α or IL-1β deficient mice were injected with LPS/GalN. Blood and liver tissue were collected at different time points, FHF related pathways were examined.ResultsAfter FHF induction the survival of both IL-1α and IL-1β KO mice was longer than that of WT mice. Lower serum liver enzyme levels, demonstrated reduced hepatic injury in the IL-1α and IL-1βKO mice. Histologically detected liver injury and apoptotic hepatocytes were significantly reduced in the IL-1αand IL-1βKO mice compared to WT mice. Reduced hepatic IkB levels and upregulated NFκB activity in WT mice remained inhibited in IL-1α and IL-1β KO mice. Hepatic expression levels of TNFα and IL-6 were significantly increased in WT mice but not in IL-1α and IL-1β KO mice.ConclusionsIL-1α and IL-1β play a central role in the pathogenesis of LPS/GalN-induced FHF. These interleukins are associated with the activation of NFκB signaling, upregulation of the pro-inflammatory cytokines and liver damage and apoptosis. Since neither IL-1α nor IL-1β depletions completely rescued the phenotype, we believe that IL-1α and IL-1β have a similar and probably complementary role in FHF progression.

Macrophage recruitment, but not interleukin 1 beta activation, enhances noise-induced hearing damage

It has been suggested that macrophages or inflammatory monocytes participate in the pathology of noise-induced hearing loss (NIHL), but it is unclear how extensively these cells contribute to the development of temporary and/or permanent NIHL. To address this question, we used clodronate liposomes to deplete macrophages and monocytes. After clodronate liposome injection, mice were exposed to 4-kHz octave band noise at 121 dB for 4 h. Compared to vehicle-injected controls, clodronate-treated mice exhibited significantly reduced permanent threshold shifts at 4 and 8 kHz and significantly smaller outer hair cell losses in the lower-apical cochlear turn. Following noise exposure, the stria vascularis had significantly more cells expressing the macrophage-specific protein F4/80, and this effect was significantly suppressed by clodronate treatment. These F4/80-positive cells expressed interleukin 1 beta (IL-1β), which noise exposure activated. However, IL-1β deficient mice did not exhibit significant resistance to intense noise when compared to wild-type mice.These findings suggest that macrophages that enter the cochlea after noise exposure are involved in NIHL, whereas IL-1β inhibition does not reverse this cochlear damage. Therefore, macrophages may be a promising therapeutic target in human sensorineural hearing losses such as NIHL.

New Insights into the Role of IL-1β in Experimental Autoimmune Encephalomyelitis and Multiple Sclerosis

Multiple sclerosis (MS), and its animal model experimental autoimmune encephalomyelitis, are neuroinflammatory diseases driven by autoreactive pathogenic TH cells that elicit demyelination and axonal damage. How TH cells acquire pathogenicity and communicate with myeloid cells and cells of the CNS remain unclear. IL-1β is recognized to play an important role in experimental autoimmune encephalomyelitis (EAE) and perhaps MS. Clinical EAE is significantly attenuated in IL-1R-deficient and IL-1β-deficient mice, and IL-1β is found in the blood, cerebrospinal fluid, and CNS lesions of MS patients. In this article, we focus on new reports that elucidate the cellular sources of IL-1β and its actions during EAE, in both lymphoid tissues and within the CNS. Several immune cell types serve as critical producers of IL-1β during EAE, with this cytokine inducing response in both hematopoietic and nonhematopoietic cells. These findings from the EAE model should inspire efforts toward investigating the therapeutic potential of IL-1 blockade in MS.

Distinctive effects of eicosapentaenoic and docosahexaenoic acids in regulating neural stem cell fate are mediated via endocannabinoid signalling pathways

Emerging evidence suggests a complex interplay between the endocannabinoid system, omega-3 fatty acids and the immune system in the promotion of brain self-repair. However, it is unknown if all omega-3 fatty acids elicit similar effects on adult neurogenesis and if such effects are mediated or regulated by interactions with the endocannabinoid system. This study investigated the effects of DHA and EPA on neural stem cell (NSC) fate and the role of the endocannabinoid signalling pathways in these effects.EPA, but not DHA, significantly increased proliferation of NSCs compared to controls, an effect associated with enhanced levels of the endocannabinoid 2-arachidonylglycerol (2-AG) and p-p38 MAPK, effects attenuated by pre-treatment with CB1 (AM251) or CB2 (AM630) receptor antagonists. Furthermore, in NSCs derived from IL-1β deficient mice, EPA significantly decreased proliferation and p-p38 MAPK levels compared to controls, suggesting a key role for IL-1β signalling in the effects observed. Although DHA similarly increased 2-AG levels in wild-type NSCs, there was no concomitant increase in proliferation or p-p38 MAPK activity. In addition, in NSCs from IL-1β deficient mice, DHA significantly increased proliferation without effects on p-P38 MAPK, suggesting effects of DHA are mediated via alternative signalling pathways. These results provide crucial new insights into the divergent effects of EPA and DHA in regulating NSC proliferation and the pathways involved, and highlight the therapeutic potential of their interplay with endocannabinoid signalling in brain repair.

IL-1β in eosinophil-mediated small intestinal homeostasis and IgA production.

Eosinophils are multifunctional leukocytes that reside in the gastrointestinal (GI) lamina propria, where their basal function remains largely unexplored. In this study, by examining mice with a selective deficiency of systemic eosinophils (by lineage ablation) or GI eosinophils (eotaxin-1/2 double–deficient or CC chemokine receptor 3–deficient), we show that eosinophils support immunoglobulin A (IgA) class switching, maintain intestinal mucus secretions, affect intestinal microbial composition, and promote the development of Peyer’s patches. Eosinophil-deficient mice showed reduced expression of mediators of secretory IgA production, including intestinal interleukin 1β (IL-1β), inducible nitric oxide synthase, lymphotoxin (LT) α, and LT-β, and reduced levels of retinoic acid-related orphan receptor gamma t–positive (ROR-γt+) innate lymphoid cells (ILCs) while maintaining normal levels of APRIL (a proliferation-inducing ligand), BAFF (B cell–activating factor of the tumor necrosis factor family), and TGF-β (transforming growth factor β). GI eosinophils expressed a relatively high level of IL-1β, and IL-1β–deficient mice manifested the altered gene expression profiles observed in eosinophil-deficient mice and decreased levels of IgA+ cells and ROR-γt+ ILCs. On the basis of these collective data, we propose that eosinophils are required for homeostatic intestinal immune responses including IgA production and that their affect is mediated via IL-1β in the small intestine.

Abstract B59: The dual effect of therapy-induced IL-1β expression on tumor progression: Role of tumor-associated macrophages

Abstract A major obstacle in clinical oncology is that tumors eventually acquire resistance to therapy. The mechanism by which resistance arises is usually associated with changes made in tumor cells in response to the therapy, however, little is known about the effect of the host which may also contribute to tumor resistance. Previous studies from our lab suggest that the induction of host cellular and molecular events following therapy may promote tumor re-growth, angiogenesis and metastasis. In this study we uncovered the possible role IL-1β - a proinflammatory cytokine - has on the growth of primary tumor and its impact on metastasis following paclitaxel (PTX) chemotherapy. We found that plasma and spleen from PTX-treated mice exhibited elevated expression of IL-1β. Furthermore, plasma from PTX-treated mice induced tumor cell invasion, an effect which was abrogated in plasma from mice treated with PTX and Anakinra, an IL-1 receptor antagonist. Consequently, mice inoculated with Lewis lung carcinoma (LLC) cells pre-exposed to plasma from mice treated with the combined therapy survived longer than mice inoculated with tumor cells pre-exposed to plasma from mice treated with PTX or Anakinra monotherapy. We next determined whether treatment efficacy of PTX is enhanced by the combination of PTX and Anakinra using 4T1 metastatic breast cancer model in mice. Tumors treated with the combined therapy were significantly smaller than tumors treated with PTX or Anakinra monotherapy. Surprisingly, although the primary tumors were smaller, such mice succumb to metastasis earlier than mice in any of the other treated groups. Furthermore, an increased number of M2 macrophages colonizing 4T1 primary tumors treated with the combined therapy was observed when compared to their number in PTX treated tumors, indicating a possible pro-metastatic role of M2 macrophages in post treatment IL-1β deprived microenvironment. Parallel results were observed when LLC bearing IL-1β deficient mice, were treated with PTX. Taken together, these findings demonstrate that although treatment outcome is enhanced following the blockade of host-induced IL1β at the primary tumor level, at the metastatic level it may serve as a double-edge sward and can lead to metastatic spread. Citation Format: Tali Voloshin, Dror Alishekevitz, Valeria Miller, Lika Isakov, Rotem Bril, Moran Benhar, Roney Apte, Yuval Shaked. The dual effect of therapy-induced IL-1β expression on tumor progression: Role of tumor-associated macrophages. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B59.

Isoflurane postconditioning reduces ischemia-induced nuclear factor-κB activation and interleukin 1β production to provide neuroprotection in rats and mice

Application of isoflurane, a volatile anesthetic, after brain ischemia can reduce ischemic brain injury in rodents (isoflurane postconditioning). This study is designed to determine whether isoflurane postconditioning improves long-term neurological outcome after focal brain ischemia and whether this protection is mediated by attenuating neuroinflammation. Adult male Sprague–Dawley rats were subjected to a 90-min middle cerebral arterial occlusion (MCAO). Isoflurane postconditioning was performed by exposing rats to 2% isoflurane for 60min immediately after the MCAO. Isoflurane postconditioning reduced brain infarct volumes, apoptotic cells in the ischemic penumbral brain tissues and neurological deficits of rats at 4weeks after the MCAO. Isoflurane postconditioning reduced brain ischemia/reperfusion-induced nuclear transcription factor (NF)-κB (NF-κB) activation as well as interleukin 1β (IL-1β) and interleukin-6 production in the ischemic penumbral brain tissues at 24h after the MCAO. IL-1β deficient mice had smaller brain infarct volumes and better neurological functions than wild-type mice at 24h after a 90-min focal brain ischemia. Isoflurane posttreatment failed to induce neuroprotection in the IL-1β deficient mice. Our results suggest that isoflurane postconditioning improved long-term neurological outcome after transient focal brain ischemia. This protection may be mediated by inhibiting NF-κB activation and the production of the proinflammatory cytokine IL-1β.

Isoflurane Induces Learning Impairment That Is Mediated by Interleukin 1β in Rodents

Postoperative cognitive decline is a clinical syndrome. Volatile anesthetics are commonly used during surgery. It is conceivable that volatile anesthetics may contribute to postoperative cognitive decline. Isoflurane can impair cognitive functions of animals under certain conditions. However, the mechanisms for this impairment are not clear. Here, male 18-month old Fisher 344 rats or 10-week old mice were exposed to 1.2 or 1.4% isoflurane for 2 h. Our studies showed that isoflurane impaired the cognitive functions of the rats in Barnes maze. Isoflurane-exposed rats had reduced freezing behavior during the training sessions in the fear conditioning test. This isoflurane effect was attenuated by lidocaine, a local anesthetic with anti-inflammatory property. Rats that had training sessions and were exposed to isoflurane 30 min later had freezing behavior similar to that of control animals. Isoflurane increased the expression of interleukin 1β (IL-1β), interleukin-6 and activated caspase 3 in the hippocampus of the 18-month old rats. IL-1β positive staining was co-localized with that of NeuN, a neuronal marker. The increase of IL-1β and activated caspase 3 but not interleukin-6 was attenuated by lidocaine. Isoflurane also impaired the cognitive functions of 10-week old C57BL/6J mice and increased IL-1β in their hippocampi. However, isoflurane did not affect the cognitive functions of IL-1β deficient mice. Our results suggest that isoflurane impairs the learning but may not affect the recall of the aged rats. IL-1β may play an important role in this isoflurane effect.

Inflammasome activation and IL-1β target IL-1α for secretion as opposed to surface expression

Interleukin-1α (IL-1α) and -β both bind to the same IL-1 receptor (IL-1R) and are potent proinflammatory cytokines. Production of proinflammatory (pro)-IL-1α and pro-IL-1β is induced by Toll-like receptor (TLR)-mediated NF-κB activation. Additional stimulus involving activation of the inflammasome and caspase-1 is required for proteolytic cleavage and secretion of mature IL-1β. The regulation of IL-1α maturation and secretion, however, remains elusive. IL-1α exists as a cell surface-associated form and as a mature secreted form. Here we show that both forms of IL-1α, the surface and secreted form, are differentially regulated. Surface IL-1α requires NF-κB activation only, whereas secretion of mature IL-1α requires additional activation of the inflammasome and caspase-1. Surprisingly, secretion of IL-1α also required the presence of IL-1β, as demonstrated in IL-1β-deficient mice. We further demonstrate that IL-1β directly binds IL-1α, thus identifying IL-1β as a shuttle for another proinflammatory cytokine. These results have direct impact on selective treatment modalities of inflammatory diseases.

Cutting Edge: Caspase-1 Independent IL-1β Production Is Critical for Host Resistance to Mycobacterium tuberculosis and Does Not Require TLR Signaling In Vivo

To investigate the respective contributions of TLR versus IL-1R mediated signals in MyD88 dependent control of Mycobacterium tuberculosis, we compared the outcome of M. tuberculosis infection in MyD88, TRIF/MyD88, IL-1R1, and IL-1beta-deficient mice. All four strains displayed acute mortality with highly increased pulmonary bacterial burden suggesting a major role for IL-1beta signaling in determining the MyD88 dependent phenotype. Unexpectedly, the infected MyD88 and TRIF/MyD88-deficient mice, rather than being defective in IL-1beta expression, displayed increased cytokine levels relative to wild-type animals. Similarly, infected mice deficient in caspase-1 and ASC, which have critical functions in inflammasome-mediated IL-1beta maturation, showed unimpaired IL-1beta production and importantly, were considerably less susceptible to infection than IL-1beta deficient mice. Together our findings reveal a major role for IL-1beta in host resistance to M. tuberculosis and indicate that during this infection the cytokine can be generated by a mechanism that does not require TLR signaling or caspase-1.

Caspase-1 independent IL-1β Production is critical for MyD88-mediated host resistance to Mycobacterium tuberculosis (42.13)

Abstract Host resistance to Mycobacterium tuberculosis (Mtb) requires the TLR/IL-1R adaptor MyD88. To investigate the contributions of TLR versus IL-1R mediated signals in MyD88 dependent control of Mtb, we compared the outcome of Mtb infection in MyD88, TRIF/MyD88, IL-1R1 and IL-1β-deficient mice. All four strains displayed acute mortality with highly increased pulmonary bacterial burdens and necrotic lung pathology suggesting a major role for IL-1β signaling in determining the MyD88 dependent phenotype. Unexpectedly, the infected MyD88 and TRIF/MyD88-deficient mice, rather than being defective in IL-1β expression, displayed increased cytokine levels relative to WT animals, arguing that MyD88 regulates host resistance to Mtb at the level of IL-1β/IL-1R1 signaling rather than TLR-mediated triggering of cytokine production. Similarly, infected mice deficient in caspase-1 and ASC, which have critical functions in inflammasome mediated IL-1β processing, showed unimpaired IL-1β production and importantly, were considerably less susceptible to infection than IL-1β deficient mice. Thus, our data argue that the production of mature host-protective IL-1β during Mtb infection can occur independently of caspase-1 activation and ASC-containing inflammasomes. Together our findings reveal a critical role for IL-1β in acute host resistance to Mtb and indicate that during this bacterial infection the cytokine can be generated by a mechanism that does not require TLR signaling or caspase-1 cleavage.

Pressure-Mediated Hypertrophy and Mechanical Stretch Induces IL-1 Release and Subsequent IGF-1 Generation to Maintain Compensative Hypertrophy by Affecting Akt and JNK Pathways

It has been reported that interleukin (IL)-1 is associated with pathological cardiac remodeling and LV dilatation, whereas IL-1beta has also been shown to induce cardiomyocyte hypertrophy. Thus, the role of IL-1 in the heart remains to be determined. We studied the role of hypertrophy signal-mediated IL-1beta/insulin-like growth factor (IGF)-1 production in regulating the progression from compensative pressure-mediated hypertrophy to heart failure. Pressure overload was performed by aortic banding in IL-1beta-deficient mice. Primarily cultured cardiac fibroblasts (CFs) and cardiac myocytes (CMs) were exposed to cyclic stretch. Heart weight, myocyte size, and left ventricular ejection fraction were significantly lower in IL-1beta-deficient mice (20%, 23% and 27%, respectively) than in the wild type 30 days after aortic banding, whereas interstitial fibrosis was markedly augmented. DNA microarray analysis revealed that IGF-1 mRNA level was markedly (approximately 50%) decreased in the IL-1beta-deficient hypertrophied heart. Stretch of CFs, rather than CMs, abundantly induced the generation of IL-1beta and IGF-1, whereas such IGF-1 induction was markedly decreased in IL-1beta-deficient CFs. IL-1beta released by stretch is at a low level unable to induce IL-6 but sufficient to stimulate IGF-1 production. Promoter analysis showed that stretch-mediated IL-1beta activates JAK/STAT to transcriptionally regulate the IGF-1 gene. IL-1beta deficiency markedly increased c-Jun N-terminal kinase (JNK) and caspase-3 activities and enhanced myocyte apoptosis and fibrosis, whereas replacement of IGF-1 or JNK inhibitor restored them. We demonstrate for the first time that pressure-mediated hypertrophy and mechanical stretch generates a subinflammatory low level of IL-1beta, which constitutively causes IGF-1 production to maintain adaptable compensation hypertrophy and inhibit interstitial fibrosis.

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.

Differential role and tissue specificity of interleukin-1α gene expression in atherogenesis and lipid metabolism

Objective We examined the role of IL-1α and IL-1β expressed by bone marrow-derived cells in atherogenesis and lipid metabolism. Methods and results We first studied the effect of atherogenic diet on wild-type C57BL/6 IL-1α or IL-1β deficient mice. IL-1α KO resulted in a comparatively higher total cholesterol levels, compared to WT and IL-1β KO mice (398 ± 10; 266 ± 19; 223 ± 13 mg/dl, respectively, p < 0.001), due to higher non-HDL cholesterol. Nevertheless, aortic sinus lesion area was 56% lower in IL-1α KO ( p < 0.05) and 50% lower in IL-1β KO ( p = 0.08), compared to WT mice. Likewise, SAA levels in IL-1α KO mice were markedly lower compared to WT and IL-1β KO mice (31 ± 14; 220 ± 33 and 106 ± 39 μg/ml, respectively, p < 0.001). To study the specific role of bone marrow-derived IL-1, irradiated C57BL/6 mice were transplanted with either IL-1+/+, IL-1α−/− or IL-1β−/− bone marrow cells. Despite similar lipoprotein levels, aortic sinus lesion area was 59% lower in IL-1α−/− transplanted ( p < 0.05) compared to IL-1+/+ transplanted mice. Lesion area in IL-1β -/- was 33% lower than in IL-1+/+ recipient mice, but it was not statistically significant. Conclusion We demonstrated that early lesion formation is accelerated specifically by bone marrow-derived IL-1α. Furthermore, we showed that the expression of IL-1α in cells other than the bone marrow plays a significant role in non-HDL cholesterol metabolism.

Critical Roles of Muscle-Secreted Angiogenic Factors in Therapeutic Neovascularization

The discovery of bone marrow-derived endothelial progenitors in the peripheral blood has promoted intensive studies on the potential of cell therapy for various human diseases. Accumulating evidence has suggested that implantation of bone marrow mononuclear cells effectively promotes neovascularization in ischemic tissues. It has also been reported that the implanted cells are incorporated not only into the newly formed vessels but also secrete angiogenic factors. However, the mechanism by which cell therapy improves tissue ischemia remains obscure. We enrolled 29 "no-option" patients with critical limb ischemia and treated ischemic limbs by implantation of peripheral mononuclear cells. Cell therapy using peripheral mononuclear cells was very effective for the treatment of limb ischemia, and its efficacy was associated with increases in the plasma levels of angiogenic factors, in particular interleukin-1beta (IL-1beta). We then examined an experimental model of limb ischemia using IL-1beta-deficient mice. Implantation of IL-1beta-deficient mononuclear cells improved tissue ischemia as efficiently as that of wild-type cells. Both wild-type and IL-1beta-deficient mononuclear cells increased expression of IL-1beta and thus induced angiogenic factors in muscle cells of ischemic limbs to a similar extent. In contrast, inability of muscle cells to secrete IL-1beta markedly reduces induction of angiogenic factors and impairs neovascularization by cell implantation. Implanted cells do not secret angiogenic factors sufficient for neovascularization but, instead, stimulate muscle cells to produce angiogenic factors, thereby promoting neovascularization in ischemic tissues. Further studies will allow us to develop more effective treatments for ischemic vascular disease.

Interleukin-18 induces production of proinflammatory cytokines in mice: no intermediate role for the cytokines of the tumor necrosis factor family and interleukin-1beta.

Interleukin-18 (IL-18) is not only a co-stimulus for the induction of interferon-gamma but also has direct proinflammatory effects by inducing tumor necrosis factor-alpha (TNF-alpha), IL-1, IL-8 and IL-6. However, the cascade of events leading to induction of cytokines by IL-18 is unclear. The aim of the present study was to investigate whether murine IL-18 stimulates production of proinflammatory cytokines, and to assess whether induction of second-wave cytokines such as IL-6 by IL-18 is driven by intermediary induction of endogenous cytokines of the TNF family or IL-1beta. When mouse peritoneal macrophages were stimulated in vitro with recombinant murine IL-18, there was a dose-dependent induction of TNF, IL-1alpha, and IL-1beta. IL-6 synthesis was also strongly induced by IL-18 and, as revealed by studies in knockout mice, this production was not dependent on interactions between endogenous cytokines of the TNF/TNF receptor family: TNF-alpha, lymphotoxin-alpha, Fas/Fas ligand (L) or CD40/CD40L. Moreover, the induction of IL-6 was also independent of endogenous IL-1beta, as macrophages isolated from IL-1beta deficient mice produced normal amounts of IL-6 after stimulation with IL-18. In conclusion, murine IL-18 has pleiotropic proinflammatory activities by inducing production of TNF-alpha, IL-1alpha, IL-1beta and IL-6, which could have important consequences for the pathophysiology of infectious and autoimmune diseases.

Hyperresponsive febrile reactions to interleukin (IL) 1alpha and IL-1beta, and altered brain cytokine mRNA and serum cytokine levels, in IL-1beta-deficient mice.

IL-1beta is an endogenous pyrogen that is induced during systemic lipopolysaccharide (LPS)- or IL-1-induced fever. We have examined the fever and cytokine responses following i.p. injection of IL-1 agonists, IL-1alpha and IL-1beta, and compared these with response to LPS (i.p.) in wild-type and IL-1beta-deficient mice. The IL-1beta deficient mice appear to have elevated body temperature but exhibit a normal circadian temperature cycle. Exogenously injected IL-1beta, IL-1alpha, or LPS induced hyperresponsive fevers in the IL-1beta-deficient mice. We also observed phenotypic differences between wild-type and IL-1beta-deficient mice in hypothalamic basal mRNA levels for IL-1alpha and IL-6, but not for IL-1beta-converting enzyme or IL-1 receptor type I or type II. The IL-1alpha mRNA levels were down-regulated, whereas the IL-6 mRNA levels were up-regulated in the hypothalamus of IL-1beta-deficient mice as compared with wild-type mice. The IL-1beta-deficient mice also responded to LPS challenge with significantly higher serum corticosterone and with lower serum tumor necrosis factor type alpha levels than the wild-type mice. The data suggest that, in the redundant cascade of proinflammatory cytokines, IL-1beta plays an important but not obligatory role in fever induction by LPS or IL-1alpha, as well as in the induction of serum tumor necrosis factor type alpha and corticosterone responses either by LPS or by IL-1alpha or IL-1beta.