A comparative study of the inhibitory effects of interleukin-1 receptor antagonist following administration as a recombinant protein or by gene transfer

Anakinra, the recombinant form of IL-1 receptor antagonist (IL-1Ra), has been approved for clinical use in the treatment of rheumatoid arthritis as the drug Kineret™, but it must be administered daily by subcutaneous injection. Gene transfer may offer a more effective means of delivery. In this study, using prostaglandin E₂ production as a measure of stimulation, we quantitatively compared the ability of anakinra, as well as that of IL-1Ra delivered by gene transfer, to inhibit the biologic actions of IL-1β. Human synovial fibroblast cultures were incubated with a range of doses of anakinra or HIG-82 cells genetically modified to constitutively express IL-1Ra. The cultures were then challenged with recombinant human IL-1β either simultaneously with addition of the source of IL-1Ra or 24 hours later. In a similar manner, the potencies of the two sources of IL-1Ra were compared when human synovial fibroblasts were challenged with IL-1β produced constitutively by genetically modified cells. No significant difference in inhibitory activity was observed between recombinant protein and IL-1Ra provided by the genetically modified cells, under static culture conditions, even following incubation for 4 days. However, under culture conditions that provided progressive dilution of the culture media, striking differences between these methods of protein delivery became readily apparent. Constitutive synthesis of IL-1Ra by the genetically modified cells provided sustained or increased protection from IL-1 stimulation over time, whereas the recombinant protein became progressively less effective. This was particularly evident under conditions of continuous IL-1β synthesis.     

Production of intracellular IL-1alpha, IL-1beta, and IL-1Ra isoforms by activated human dermal and synovial fibroblasts: phenotypic differences between human dermal and synovial fibroblasts

We compared the production of IL-1alpha, IL-1beta, and of IL-1Ra isoforms by cultured human dermal (HDF) and synovial fibroblasts (HSF) in response to IL-1alpha, TNF-alpha, or direct T cell membrane contact. IL-1Ra was constitutively present in the cell lysates of cultured HDF and its synthesis increased in stimulated cells, whereas IL-1Ra was present in low amounts in the supernatants. Secreted IL-1Ra (sIL-1Ra) and intracellular IL-1Ra type 1 (icIL-1Ra1) mRNA levels followed the same pattern. In stimulated HDF, IL-1alpha and IL-1beta were increased intracellularly but remained undetectable in the supernatants. In HSF, IL-1Ra levels increased in both cell lysates and supernatants upon stimulation. IL-1beta was only present in HSF cell lysates after stimulation, whereas IL-1alpha was undetectable. Both sIL-1Ra and icIL-1Ra1 mRNAs were detected in stimulated HSF. icIL-1Ra1 was the predominant intracellular isoform in both cell types. In conclusion, stimulated HDF produce high amounts of intracellular IL-1Ra, IL-1alpha, and IL-1beta. In contrast, HSF synthesized both intracellular and secreted IL-1Ra, whereas IL-1beta was present only in cell lysates. The presence of high amounts of icIL-1Ra1 and intracellular IL-1alpha in HDF suggests that these cytokines may carry out important function inside cells.     

Production of interleukin-1 receptor antagonist isoforms by microglia in mixed rat glial cells stimulated by lipopolysaccharide

Although the natural interleukin-1 receptor antagonist (IL-1Ra) has been shown to be produced by microglial cells in response to immune stimuli, nothing was known about the ability of these cells in primary culture to produce the different isoforms of IL-1Ra. Using RT-PCR, we first confirmed that mixed glial cell cultures from newborn rats respond to the cytokine inducer, lipopolysaccharide, by synthesizing IL-1Ra mRNA. Using double immunostaining, we showed that IL-1Ra was detected in microglia but not in astrocytes. Using Western blotting, we finally demonstrated that the IL-1Ra1 isoform was secreted in the supernatant of mixed glial cell cultures, and its production increased in response to lipopolysaccharide. The three different IL-1Ra isoforms were constitutively expressed in cell lysates and their levels increased after lipopolysaccharide treatment, except for IL-1Ra3. These results point to the ability of microglial cells in primary culture to produce the different isoforms of IL-1Ra.     

IL-1 receptor antagonist regulation of acute phase protein synthesis in human hepatoma cells.

The hepatoma cell line HuH-7 has recently been shown to synthesize serum amyloid A (SAA) in response to IL-1. IL-1 receptor antagonist (IL-1Ra) was able to completely inhibit the response of SAA to IL-1 but not the increase seen in response to IL-6. IL-1Ra was equally effective at inhibiting IL-1 alpha or IL-1 beta. At a 10-fold molar excess of IL-1Ra over IL-1 there was complete inhibition of the SAA response. Removal of IL-1 at 24 h rapidly reduced the SAA secreted over the next 24 h. Addition of IL-1Ra to the cells at this time was as effective as removal of IL-1 at inhibiting the subsequent secretion of SAA. IL-1Ra was less effective at inhibition of IL-1-induced haptoglobin secretion. We would conclude that IL-1Ra may play an important role in the regulation of acute phase protein synthesis.     

The active metabolite of leflunomide,A77 1726, increases the production of IL-1 receptor antagonist in human synovial fibroblasts and articular chondrocytes

Leflunomide is an immunomodulatory agent used for the treatment of rheumatoid arthritis. In this study, we investigated the effect of A77 1726 – the active metabolite of leflunomide – on the production of IL-1 receptor antagonist (IL-1Ra) by human synovial fibroblasts and articular chondrocytes. Cells were incubated with A77 1726 alone or in combination with proinflammatory cytokines. IL-1Ra production was determined by ELISA. A77 1726 alone had no effect, but in the presence of IL-1β or tumour necrosis factor-α it markedly enhanced the secretion of IL-1Ra in synovial fibroblasts and chondrocytes. The effect of A77 1726 was greatest at 100 μmol/l. In synovial fibroblasts and de-differentiated chondrocytes, A77 1726 also increased IL-1β-induced IL-1Ra production in cell lysates. Freshly isolated chondrocytes contained no significant amounts of intracellular IL-1Ra. A77 1726 is a known inhibitor of pyrimidine synthesis and cyclo-oxygenase (COX)-2 activity. Addition of exogenous uridine did not significantly modify the effect of A77 1726 on IL-1Ra production, suggesting that it was not mediated by inhibition of pyrimidine synthesis. Indomethacin increased IL-1β-induced IL-1Ra secretion in synovial fibroblasts and de-differentiated chondrocytes, suggesting that inhibition of COX-2 may indeed enhance IL-1β-induced IL-1Ra production. However, the stimulatory effect of indomethacin was consistently less effective than that of A77 1726. A77 1726 increases IL-1Ra production by synovial fibroblasts and chondrocytes in the presence of proinflammatory cytokines, and thus it may possess chondroprotective effects. The effect of A77 1726 may be partially mediated by inhibition of COX-2, but other mechanisms likely concur to stimulate IL-1Ra production.     

An anti-inflammatory property of Candida albicans β-glucan: Induction of high levels of interleukin-1 receptor antagonist via a Dectin-1/CR3 independent mechanism

BackgroundCandida albicans is an opportunistic fungal pathogen that induces strong proinflammatory responses, such as IL-1β production. Much less is known about the induction of immune modulatory cytokines, such as the IL-1 receptor antagonist (IL-1Ra) that is the main natural antagonist of IL-1, by C. albicans.MethodsPeripheral blood mononuclear cells (PBMC) of healthy individuals were stimulated with C. albicans and different components of the fungal cell wall. The role of pathogen recognition receptors (PRRs) for the induction of IL-1β and IL-1Ra was investigated by using specific blockers or in PBMC from Dectin-1 deficient patients.ResultsC. albicans induced a strong IL-1Ra response, and this induction was primarily induced by the cell-wall component β-glucan. Blocking IL-1Ra significantly increased C. albicans β-glucan hyphae induced IL-1β and IL-6 production. Surprisingly, blocking the β-glucan receptor Dectin-1 or the downstream Syk or Raf-1 pathways only marginally reduced C. albicans-induced IL-1Ra production, while blocking of the complement receptor 3 (CR3), TLR2 or TLR4 had no effect. In line with this, blocking MAP kinases had little effect on Candida-induced IL-1Ra production. PBMC isolated from Dectin-1 deficient patients produced normal IL-1Ra amounts in response to C. albicans stimulation. Interestingly, the IL-1Ra synthesis induced by β-glucan was blocked by inhibitors of the Akt/PI3 K pathway.Conclusionsβ-glucan of C. albicans induces a strong IL-1Ra response, which is independent of the β-glucan receptors dectin-1 and CR3. These data strongly argue for the existence of an unknown β-glucan receptor that specifically induces an Akt/PI3 K-dependent anti-inflammatory IL-1Ra response upon recognition of C. albicans.     

Interleukin 1 components in cicatricial pemphigoid. Role in intravenous immunoglobulin therapy.

Interleukin (IL-)1 is an important mediator of inflammatory responses and plays an important role in the pathogenesis of various autoimmune diseases. Cicatricial pemphigoid (CP) is a multisystem autoimmune inflammatory disease. We have studied the role of IL-1 in its pathogenesis. We have investigated the serum levels of IL-1 components (IL-1alpha, IL-1beta, and IL-1Ra), and determined the role of intravenous immunoglobulin (IVIg) therapy in patients with CP. Serum levels of IL-1alpha and beta were significantly higher in untreated patients with active disease compared to levels in patients in prolonged clinical remission and normal human controls (P<0.0001). The serum levels of IL-1Ra were higher in patients in prolonged clinical remission compared to patients with active disease (P=0.002). Hence elevated levels of IL-1alpha and beta and low levels of IL-1Ra correlate with disease activity. The levels of IL-1alpha and beta were statistically significantly higher in sera of CP patients with active disease pre-IVIg therapy compared to post-IVIg therapy (P<0.0001). Statistically significantly higher levels of IL-1Ra were present in post-IVIg treatment serum samples when compared to levels in pre-IVIg treatment (P<0.0001). In the in vitro experiments, the levels of IL-1alpha and beta produced by the peripheral blood mononuclear cells (PBMC) isolated from patients before IVIg therapy were significantly higher when compared to the PBMC isolated from post-IVIg patients (P<0.0001). Significantly higher levels of IL-1Ra were observed in the supernatants of PBMC collected from pre-IVIg patients and cultured with exogenously added IVIg, when compared to the levels of PBMC to which IVIg was not added (P<0.0001). IL-1 may be an important cytokine involved in the pathogenesis of CP. The regulation of IL-1 could be one of the mechanisms, amongst others, by which IVIg may exert its beneficial effect in the treatment of CP.     

Imbalance production between interleukin-1beta (IL-1beta) and IL-1 receptor antagonist (IL-1Ra) in bronchial asthma

Genes of the IL-1 family encode three different peptides, IL-1alpha, IL-1beta, and IL-1Ra, respectively. IL-1 operates through IL-1RI, and is involved in airway inflammation in asthmatic subjects, whereas IL-1Ra appears to be a specific competitive inhibitor of IL-1. All genes are on chromosome 2q12-21 where genomewide searches have identified linkage for asthma. To test whether variants of IL-1 relate to asthma, we conducted a genetic association study in a Japanese population. We show that the A2 allele of IL1RN (encoding IL-1Ra) associates with nonatopic asthma [OR = 5.71, 95% CI: 1.63-19. 8, Pc = 0.007]. Both atopic and nonatopic asthmatics with the A2 allele had significantly lower serum IL-1Ra levels in both types of asthmatics. Peripheral blood cells from asthmatics with A2 alleles, however, produced as much IL-1 as did those with A1 homozygotes. Since Th1 and Th2 cytokines differentially regulate the ratio between IL-1beta and IL-1Ra, these findings suggest that dysregulation of IL-1beta/IL-1Ra, probably due to interaction between epithelium and immuno-competent cells in the airway, is important in asthma inflammation.     

Intracellular IL-1Ra type 1 inhibits IL-1-induced IL-6 and IL-8 production in Caco-2 intestinal epithelial cells through inhibition of p38 mitogen-activated protein kinase and NF-kappaB pathways

Interleukin-1 (IL-1) plays a pivotal role in the pathogenesis of inflammatory bowel disease (IBD). IL-1 action is regulated in part by its naturally occurring inhibitor, the IL-1 receptor antagonist (IL-1Ra). Four splice variants of IL-1Ra gene product have been described, one secreted (sIL-1Ra) and three intracellular (icIL-1Ra1, 2, 3). Although sIL-1Ra and icIL-1Ra1 bind to type I IL-1 receptor with equal affinity, icIL-1Ra1 may carry out unique functions inside cells. The goal of this study was to determine the role of icIL-1Ra1 in regulation of cytokine-induced IL-6 and IL-8 production in Caco-2 intestinal epithelial cells. icIL-1Ra1 inhibited IL-1-induced IL-6 and IL-8 production. IL-1 activated all three mitogen-activated protein (MAP) kinase family members: p38 MAP kinase, extracellular-regulated kinases (ERK), and c-Jun amino-terminal kinases (JNK). Specific inhibitors of each MAP kinase pathway decreased IL-1-induced IL-6 and IL-8 production. Overexpression of icIL-1Ra1 inhibited p38 MAP kinase phosphorylation, but had no effect on ERK and JNK phosphorylation. In addition, icIL-1Ra1 inhibited nuclear translocation of NF-kappaB after IL-1 stimulation. In conclusion, these data indicate that icIL-1Ra1, acting in the cytoplasm of Caco-2 cells, decreased IL-1-induced IL-6 and IL-8 production. This intracellular anti-inflammatory activity of icIL-1Ra1 was mediated through inhibition of p38 MAP kinase and NF-kappaB signal transduction pathways.     

Interleukin 1 induces HIV-1 expression in chronically infected U1 cells: blockade by interleukin 1 receptor antagonist and tumor necrosis factor binding protein type 1.

BACKGROUND: Cytokines and cytokine antagonists modulate human immunodeficiency virus (HIV) replication in vitro and may be involved in HIV disease pathogenesis. An understanding of these cytokine networks may suggest novel treatment strategies for HIV-seropositive persons. MATERIALS AND METHODS: U1 cells, a chronically infected promonocytic cell line, were stimulated with interleukin 1 alpha (IL-1 alpha), IL-1 beta or tumor necrosis factor (TNF) for 24 hr. The effects of these cytokines, and of anti-IL-1 receptor type 1 and type 2 (IL-1RI and II) antibody, IL-1 receptor antagonist (IL-1Ra), and recombinant human TNF binding protein type 1 (rhTBP-1, a form of TNF receptor p55), on HIV-1 replication, as measured by ELISA for HIV-1 p24 antigen, were determined. The effects of IL-1 and IL-1Ra on nuclear factor-kappa B (NF-kappa B) DNA binding activity, as measured by electrophoretic mobility shift assays, were also determined. RESULTS: IL-1 alpha and IL-1 beta increased p24 antigen production in a concentration-dependent manner. IL-1Ra completely, and rhTBP-1 partially, suppressed IL-1-induced p24 antigen production. IL-1 increased NF-kappa B DNA binding activity and IL-1Ra blocked this effect. Since IL-1Ra blocks IL-1 from binding to both the IL-1RI and Il-1RII, monoclonal antibodies directed against each receptor were used to ascertain which IL-1R mediates IL-1-induced HIV-1 expression. Antibody to the IL-1RI reduced IL-1-induced p24 antigen production. Although anti-IL-1RII antibody blocked the binding of 125IL-1-1 alpha to U1 cells by 99%, this antibody did not affect IL-1-induced p24 antigen production. IL-1 beta enhanced TNF alpha-induced HIV expression when added before or simultaneously with TNF alpha. CONCLUSIONS: IL-1 induces HIV-1 expression (via the IL-1RI) and NF-kappa B activity in U1 cells. These effects are blocked by IL-1Ra and partially mediated by TNF. IL-1 enhances TNF alpha-induced HIV replication in U1 cells.     

The balance between IL-1 and IL-1Ra in disease

IL-1 is an important mediator of inflammation and tissue damage in multiple organs, both in experimental animal models of disease and in human diseases. The IL-1 family consists of two agonists, IL-1alpha and IL-1beta, two receptors, biologically active IL-1RI and inert IL-1RII, and a specific receptor antagonist, IL-1Ra. The balance between IL-1 and IL-1Ra in local tissues plays an important role in the susceptibility to and severity of many diseases. An allelic polymorphism in the IL-1Ra gene has been associated with a variety of human diseases primarily of epithelial and endothelial cell origin. This association may be secondary to an imbalance in the IL-1 system with enhanced production of IL-1beta and reduced production of the major intracellular isoform of IL-1Ra. Treatment of RA with daily subcutaneous injections of recombinant IL-1Ra protein has been shown to be efficacious. Gene therapy approaches with IL-1Ra are being evaluated for the treatment of RA and other human diseases.     

Adipose and liver expression of interleukin (IL)-1 family members in morbid obesity and effects of weight loss

Morbid obesity is associated with a state of chronic inflammation. Interleukin-1 family (IL-1F) cytokine members are produced by human adipose tissue in obesity. Whereas certain IL-1F members such as IL-1β or IL-18 are potently proinflammatory, others such as IL-1 receptor antagonist (IL-1Ra) or IL-37 (formerly IL-1F7) are antiinflammatory. The NLRP3 inflammasome plays a key role in the processing of bioactive IL-1β and IL-18. We investigated the effect of excessive weight loss on subcutaneous adipose tissue and liver expression of IL-1α, IL-1β, IL-18, IL-1Ra, IL-37 and NLRP3. Twenty-one severely obese patients undergoing laparoscopic adjustable gastric banding were studied. Tissue samples were collected before and 6 months after laparoscopic adjustable gastric banding surgery. mRNA expression of all studied IL-1F members, but especially of IL-37, was much higher in subcutaneous/visceral adipose tissue compared with their liver expression. Subcutaneous adipose tissue mRNA expression of IL-1β decreased significantly after extensive weight loss; expression of IL-18 and IL-1Ra did not change, whereas IL-37 expression increased. Weight loss led to a significant reduction in liver IL-1β, IL-18 and IL-1Ra expression, whereas hepatic IL-37 mRNA expression remained stable. Adipose/liver NLRP3 inflammasome and IL-1α expression were not affected by weight loss. Tissue expression of IL-1β, IL-18 and IL-37 were significantly higher in subcutaneous/visceral adipose tissue compared with the liver. In conclusion, expression of IL-1F members is more pronounced in adipose compared with liver tissue in patients with severe obesity. Excessive weight loss changes the adipose and liver expression profile of IL-1F members toward a more antiinflammatory direction.     

Intracellular interleukin-1 receptor antagonist type 1 antagonizes the stimulatory effect of interleukin-1 alpha precursor on cell motility

Interleukin (IL)-1alpha, a proinflammatory cytokine, is produced as a 33 kDa protein precursor (preIL-1alpha) which is cleaved to generate the 17 kDa C-terminal mature IL-1alpha (mIL-1alpha) and the 16kDa N-terminal IL-1alpha propiece (NIL-1alpha). The biological effect of IL-1alpha is regulated by the IL-1 receptor antagonist (IL-1Ra), its naturally occurring inhibitor. Four different isoforms of the IL-1Ra have been described, one secreted (sIL-1Ra) and three intracellular (icIL-1Ra1, 2, 3). Whether the icIL-1Ra1 isoform can antagonize some of the biological effects of intracellular IL-1alpha is still unknown. The aim of this study is to investigate effects of preIL-1alpha and icIL-1Ra1 on cell motility in stably transfected ECV304 cells. We show that expression of preIL-1alpha in ECV304 cells significantly increases cell motility. Furthermore, transfection with NIL-1alpha propiece also increases cell motility whereas this stimulatory effect was not observed by addition of exogenous mIL-1alpha, suggesting an intracellular effect of preIL-1alpha mediated by NIL-1alpha propiece. Co-transfection of ECV304 cells with icIL-1Ra1 completely antagonizes the stimulatory effect of preIL-1alpha and NIL-1alpha propiece on cell motility. In conclusion, NIL-1alpha propiece increases ECV304 cell motility and icIL-1Ra1 exerts intracellular functions regulating this stimulatory effect.     

Intracellular IL-1 receptor antagonist is elevated in human dermal fibroblasts that overexpress intracellular precursor IL-1 alpha.

Cultured dermal fibroblasts from systemic sclerosis patients express higher levels of intracellular IL-1 alpha than fibroblasts from healthy controls. In this study, we found that systemic sclerosis dermal fibroblasts also express higher levels of the intracellular isoform of IL-1 receptor antagonist (icIL-1Ra) than normal fibroblasts after stimulation with IL-1 beta or TNF-alpha. A possible relationship between elevated precursor IL-1 alpha (preIL-1 alpha) and elevated icIL-1Ra was investigated by transducing normal dermal fibroblasts to overexpress preIL-1 alpha, preIL-1 beta, or icIL-1Ra. Fibroblasts that overexpressed icIL-1Ra did not have elevated levels of IL-1 alpha. On the other hand, fibroblasts that overexpressed preIL-1 alpha had at least 4-fold higher basal levels of icIL-1Ra than control fibroblasts and 4-fold higher levels of icIL-1Ra after induction with IL-1 beta or TNF-alpha. Fibroblasts overexpressing preIL-1 beta did not exhibit elevated icIL-1Ra. The differences in icIL-1Ra protein levels were reflected in differences in mRNA. In contrast, IL-1-stimulated levels of MCP-1 and IL-6 were not different in control and preIL-1 alpha-transduced fibroblasts. Addition of neutralizing anti-IL-1 alpha Abs to fibroblast cultures did not diminish basal or stimulated levels of icIL-1Ra in the preIL-1 alpha-transduced cells, supporting an intracellular site of action of preIL-1 alpha. This is the first report of an association between intracellular levels of these IL-1 family members. We hypothesize that intracellular preIL-1 alpha participates in the regulation of icIL-1Ra.     

Interleukin Expression after Injury and the Effects of Interleukin-1 Receptor Antagonist

Ligament healing follows a series of complex coordinated events involving various cell types, cytokines, as well as other factors, producing a mechanically inferior tissue more scar-like than native tissue. Macrophages provide an ongoing source of cytokines to modulate inflammatory cell adhesion and migration as well as fibroblast proliferation. Studying interleukins inherent to ligament healing during peak macrophage activation and angiogenesis may elucidate inflammatory mediators involved in subsequent scar formation. Herein, we used a rat healing model assayed after surgical transection of their medial collateral ligaments (MCLs). On days 3 and 7 post-injury, ligaments were collected and used for microarray analysis. Of the 12 significantly modified interleukins, components of the interleukin-1 family were significantly up-regulated. We therefore examined the influence of interleukin-1 receptor antagonist (IL-1Ra) on MCL healing. Transected rat MCLs received PBS or IL-1Ra at the time of surgery. Inhibition of IL-1 activation decreased pro-inflammatory cytokines (IL-1α, IL-1β, IL-12, IL-2, and IFN-γ), myofibroblasts, and proliferating cells, as well as increased anti-inflammatory cytokines (IL-10), endothelial cells/blood vessel lumen, M2 macrophages, and granulation tissue size without compromising the mechanical properties. These results support the concept that IL-1Ra modulates MCL-localized granulation tissue components and cytokine production to create a transient environment that is less inflammatory. Overall, IL-1Ra may have therapeutic potential early in the healing cascade by stimulating the M2 macrophages and altering the granulation tissue components. However, the single dose of IL-1Ra used in this study was insufficient to maintain the more regenerative early response. Due to the transient influence on most of the healing components tested, IL-1Ra may have greater therapeutic potential with sustained delivery.     

Interleukin-1 Receptor Antagonist Has a Novel Function in the Regulation of Matrix Metalloproteinase-13 Expression

Interleukin-1 receptor antagonist (IL-1Ra) is an IL-1 family member, which binds to IL-1 receptors but does not induce any intracellular signaling. We addressed whether IL-1Ra has a novel function in regulation of the extracellular matrix or adhesion molecules. Polymerase chain reaction array analysis demonstrated a ~5-fold increase in matrix metalloproteinase 13 (MMP-13) mRNA expression of IL-1Ra siRNA-transfected Ca9-22 human oral squamous epithelial carcinoma cells compared with the control. In fact, MMP-13 mRNA and protein expression as well as its activity in IL-1Ra siRNA-transfected Ca9-22 cell lines were significantly higher than those in the control. IL-1Ra siRNA treatment resulted in strong elevation of MMP-13 expression, whereas addition of rhIL-1Ra (40 ng/ml) suppressed MMP-13 expression, suggesting that IL-1Ra had a specific effect on MMP-13 induction. IL-1Ra siRNA could potently suppress IL-1α. No significant difference was found between the MMP-13 mRNA expression of IL-1Ra siRNA-transfected cells and those treated with anti-IL-1α or anti-IL-1β antibodies. These results suggested that continuous supply of IL-1 had no effect on the induction of MMP-13 by IL-1Ra siRNA. Histopathological investigation of MMP-13 in periodontal tissue showed specific localization in the junctional epithelial cells of IL-1Ra knockout (KO) mice. Furthermore, infection with Aggregatibacter actinomycetemcomitans to establish an experimental periodontitis model resulted in predominant localization of MMP-13 along apical junctional epithelial cells. Laminin-5, which is degraded by MMP-13, was found in the internal basal lamina of wild-type mice, whereas the internal basal lamina of IL-1Ra KO mice did not show obvious laminin-5 localization. In particular, laminin-5 localization almost disappeared in the internal basal lamina of IL-1Ra KO mice infected with A. actinomycetemcomitans, suggesting that the suppression of IL-1Ra resulted in strong induction of MMP-13 that degraded laminin-5. In conclusion, IL-1Ra is associated with MMP-13 expression and has a novel function in such regulation without interference of the IL-1 signaling cascade.