X‐ray crystal structure localizes the mechanism of inhibition of an IL‐36R antagonist monoclonal antibody to interaction with Ig1 and Ig2 extra cellular domains

Cellular signaling via binding of the cytokines IL‐36α, β, and γ along with binding of the accessory protein IL‐36RAcP, to their cognate receptor IL‐36R is believed to play a major role in epithelial and immune cell‐mediated inflammation responses. Antagonizing the signaling cascade that results from these binding events via a directed monoclonal antibody provides an opportunity to suppress such immune responses. We report here the molecular structure of a complex between an extracellular portion of human IL‐36R and a Fab derived from a high affinity anti‐IL‐36R neutralizing monoclonal antibody at 2.3 Å resolution. This structure, the first of IL‐36R, reveals similarities with other structurally characterized IL‐1R family members and elucidates the molecular determinants leading to the high affinity binding of the monoclonal antibody. The structure of the complex reveals that the epitope recognized by the Fab is remote from both the putative ligand and accessory protein binding interfaces on IL‐36R, suggesting that the functional activity of the antibody is noncompetitive for these binding events.     

Blockade of IL-36 Receptor Signaling Does Not Prevent from TNF-Induced Arthritis

Introduction

Interleukin (IL)-36α is a newly described member of the IL-1 cytokine family with a known inflammatory and pathogenic function in psoriasis. Recently, we could demonstrate that the receptor (IL-36R), its ligand IL-36α and its antagonist IL-36Ra are expressed in synovial tissue of arthritis patients. Furthermore, IL-36α induces MAP-kinase and NFκB signaling in human synovial fibroblasts with subsequent expression and secretion of pro-inflammatory cytokines.

Methods

To understand the pathomechanism of IL-36 dependent inflammation, we investigated the biological impact of IL-36α signaling in the hTNFtg mouse. Also the impact on osteoclastogenesis by IL-36α was tested in murine and human osteoclast assays.

Results

Diseased mice showed an increased expression of IL-36R and IL-36α in inflamed knee joints compared to wildtype controls. However, preventively treating mice with an IL-36R blocking antibody led to no changes in clinical onset and pattern of disease. Furthermore, blockade of IL-36 signaling did not change histological signs of TNF-induced arthritis. Additionally, no alteration on bone homeostasis was observed in ex vivo murine and human osteoclast differentiation assays.

Conclusion

Thus we conclude that IL-36α does not affect the development of inflammatory arthritis.     

The Crosstalk between IL-22 Signaling and miR-197 in Human Keratinocytes

The interaction between the immune system and epithelial cells is tightly regulated. Aberrations of this balance may result in inflammatory diseases such as psoriasis, inflammatory bowel disease and rheumatoid arthritis. IL-22 is produced by Th17, Th22 and Th1 cells. Putative targets for IL-22 are cells in the skin, kidney, digestive and respiratory systems. The highest expression of IL-22 receptor is found in the skin. IL-22 plays an important role in the pathogenesis of T cell-mediated inflammatory diseases such as psoriasis, inflammatory bowel disease and rheumatoid arthritis. Recently, we found that miR-197 is down regulated in psoriatic lesions. In the present work we show that miR-197 over expression inhibits keratinocytes proliferation induced by IL-22 and keratinocytes migration. In addition, we found that IL-22 activates miR-197 expression through the binding of phosphorylated STAT3 to sequences in the putative promoter of miR-197. Finally we found that IL-22 receptor subunit IL22RA1 is a direct target of miR-197. Hence, we identified a novel feedback loop controlling IL-22 signaling, in which IL-22 induces miR-197, which in turn, negatively regulates IL-22 receptor and attenuates the biological outcome of such signaling. Regulation of this pathway may be important in inflammatory skin disorders such a psoriasis and in wound healing.     

IL-36 family cytokines in protective versus destructive inflammation

The IL-1 family of cytokines and receptors are critical regulators of inflammation. Within the IL-1 family and in contrast to its IL-1 and IL-18 subfamilies, the IL-36 subfamily is still poorly characterized. Three pro-inflammatory agonists IL-36α, IL-36β, IL-36γ, one IL-36 receptor (IL-1R6) antagonist, IL-36RA, and one putative IL-1R6 antagonist, IL-38, have been grouped into the IL-36 cytokine subfamily. IL-36 agonists signal through a common receptor complex to serve as early triggers of inflammatory responses by activating and cross-regulating a number of inflammatory pathways including NF-κB, MAPK and IFN signaling. IL-36RA binds to IL-1R6 to limit inflammatory signaling, while IL-38 may be an antagonist of more than one IL-1 family receptor. Expression patterns of IL-36 family cytokines, being most prominently expressed in epithelial barrier tissues such as the skin and intestines as well as in immune cells, suggest a role in protecting these barriers from infection. Dysregulation of IL-36 family cytokine signaling at physiological barriers, most prominently the skin, induces autoimmune inflammation. However, transferring the potential of IL-36 to induce tissue damage to tumors might benefit cancer patients. Here we summarize signaling pathways regulated by IL-36 family cytokines, including IL-38, and the consequences for physiological protective and pathophysiological destructive inflammation. Moreover, we discuss the limits of current knowledge on IL-36 family function to open potential avenues for research in the future.     

IL‐35 recombinant protein reverses inflammatory bowel disease and psoriasis through regulation of inflammatory cytokines and immune cells

Interleukin‐35 (IL‐35), a member of the IL‐12 family, functions as a new anti‐inflammatory factor involved in arthritis, psoriasis, inflammatory bowel disease (IBD) and other immune diseases. Although IL‐35 can significantly prevent the development of inflammation in many diseases, there have been no early studies accounting for the role of IL‐35 recombinant protein in IBD and psoriasis. In this study, we assessed the therapeutic potential of IL‐35 recombinant protein in three well‐known mouse models: the dextransulfate sodium (DSS)‐induced colitis mouse model, the keratin14 (K14)‐vascular endothelial growth factor A (VEGF‐A)‐transgenic (Tg) psoriasis mouse model and the imiquimod (IMQ)‐induced psoriasis mouse model. Our results indicated that IL‐35 recombinant protein can slow down the pathologic process in DSS‐induced acute colitis mouse model by decreasing the infiltrations of macrophages, CD4⁺T and CD8⁺T cells and by promoting the infiltration of Treg cells. Further analysis demonstrated that IL‐35 recombinant protein may regulate inflammation through promoting the secretion of IL‐10 and inhibiting the expression of pro‐inflammatory cytokines such as IL‐6, TNF‐α and IL‐17 in acute colitis model. In addition, lower dose of IL‐35 recombinant protein could achieve long‐term treatment effects as TNF‐α monoclonal antibody did in the psoriasis mouse. In summary, the remarkable therapeutic effects of IL‐35 recombinant protein in acute colitis and psoriasis mouse models indicated that IL‐35 recombinant protein had a variety of anti‐inflammatory effects and was expected to become an effective candidate drug for the treatment of inflammatory diseases.     

Cutting edge: A critical functional role for IL-23 in psoriasis

Interleukin-23 is a key cytokine involved in the generation of Th17 effector cells. Clinical efficacy of an anti-p40 mAb blocking both IL-12 and IL-23 and disease association with single nucleotide polymorphisms in the IL23R gene raise the question of a functional role of IL-23 in psoriasis. In this study, we provide a comprehensive analysis of IL-23 and its receptor in psoriasis and demonstrate its functional importance in a disease-relevant model system. The expression of IL-23 and its receptor was increased in the tissues of patients with psoriasis. Injection of a mAb specifically neutralizing human IL-23 showed IL-23-dependent inhibition of psoriasis development comparable to the use of anti-TNF blockers in a clinically relevant xenotransplant mouse model of psoriasis. Together, our results identify a critical functional role for IL-23 in psoriasis and provide the rationale for new treatment strategies in chronic epithelial inflammatory disorders.     

IL-36 in chronic inflammation and cancer

IL-36 belongs to the IL-1 family of cytokines and activates target cells by binding to a specific cytokine receptor (IL-36R) followed by activation of intracellular regulators such as MAP kinases and NF-kappaB. Three subforms of IL-36, denoted IL-36alpha, IL-36beta and IL-36gamma, have been described that require N-terminal cleavage for activation. Functional studies have shown that IL-36 may activate a broad spectrum of immune and non-immune cells such as macrophages, T cells, keratinocytes and epithelial cells in an IL-1-independent fashion and thereby controls various inflammatory or oncogenic processes in the skin, the lung, the kidney, the liver and the intestine, respectively. Based on the presence of mutations of the IL-36RN in patients with generalized pustular psoriasis, successful clinical pilot trials with IL-36R blocking antibodies were conducted in these patients and further studies in patients with autoimmune or chronic inflammatory disorders such as inflammatory bowel diseases are under way. Collectively, these findings highlight a crucial regulatory role of IL-36 signaling in driving various inflammatory disorders that provide a rational basis for clinical targeting of this cytokine.     

Reconstitution of ST2 (IL-1R4) specific for IL-33 activity; no suppression by IL-1Ra though a common chain IL-1R3 (IL-1RAcP) shared with IL-1

Interleukin-33 (IL-33) receptors are composed of ST2 (also known as IL-1R4), a ligand binding chain, and IL-1 receptor accessory protein (IL-1RAcP, also known as IL-1R3), a signal transducing chain. IL-1R3 is a common receptor for IL-1α, and IL-1β, IL-33, and three IL-36 isoforms. A549 human lung epithelial cells are highly sensitive to IL-1α and IL-1β but not respond to IL-33. The lack of responsiveness to IL-33 is due to ST2 expression. ST2 was stably transfected into A549 cells to reconstitute its activity. RT-PCR and FACS analysis confirmed ST2 expression on the cell surface of A549/ST2 cells. Upon IL-33 stimulation, A549/ST2 cells induced IL-8 and IL-6 production in a dose dependent manner while A549/mock cells remained unresponsive. There was no difference in IL-1α and IL-1β activity in A549/ST2 cells compared to A549/mock cells despite the fact that IL-33 shares IL-1R3 with IL-1α/β. IL-33 activated inflammatory signaling molecules in a time- and dose-dependent manner. Anti-ST2 antibody and soluble recombinant ST2-Fc abolished IL-33-induced IL-6 and IL-8 production in A549/ST2 cells but the IL-1 receptor antagonist failed to block IL-33-induced cytokines. This result demonstrates for the first time the reconstitution of ST2 in A549 human lung epithelial cell line and verified its function in IL-33-mediated cytokine production and signal transduction.     

Interleukin-36 potently stimulates human M2 macrophages,Langerhans cells and keratinocytes to produce pro-inflammatory cytokines

Interleukin (IL)-36 cytokines belong to the IL-1 family and include three agonists, IL-36 α, β and γ and one inhibitor, IL-36 receptor antagonist (IL-36Ra). IL-36 and IL-1 (α and β) activate similar intracellular pathways via their related heterodimeric receptors, IL-36R/IL-1RAcP and IL-1R1/IL-1RAcP, respectively. However, excessive IL-36 versus IL-1 signaling induces different phenotypes in humans, which may be related to differential expression of their respective receptors.We examined the expression of IL-36R, IL-1R1 and IL-1RAcP mRNA in human peripheral blood, tonsil and skin immune cells by RT-qPCR. Monocyte-derived dendritic cells (MDDC), M0, M1 or M2-polarized macrophages, primary keratinocytes, dermal macrophages and Langerhans cells (LC) were stimulated with IL-1β or IL-36β. Cytokine production was assessed by RT-qPCR and immunoassays.The highest levels of IL-36R mRNA were found in skin-derived keratinocytes, LC, dermal macrophages and dermal CD1a⁺ DC. In the blood and in tonsils, IL-36R mRNA was predominantly found in myeloid cells. By contrast, IL-1R1 mRNA was detected in almost all cell types with higher levels in tonsil and skin compared to peripheral blood immune cells. IL-36β was as potent as IL-1β in stimulating M2 macrophages, keratinocytes and LC, less potent than IL-1β in stimulating M0 macrophages and MDDC, and exerted no effects in M1 and dermal macrophages. Levels of IL-1Ra diminished the ability of M2 macrophages to respond to IL-1.Taken together, these data are consistent with the association of excessive IL-36 signaling with an inflammatory skin phenotype and identify human LC and M2 macrophages as new IL-36 target cells.     

Suppression of TNF-α and IL-1 signaling identifies a mechanism of homeostatic regulation of macrophages by IL-27

IL-27 is a pleiotropic cytokine with both activating and inhibitory functions on innate and acquired immunity. IL-27 is expressed at sites of inflammation in cytokine-driven autoimmune/inflammatory diseases, such as rheumatoid arthritis, psoriasis, inflammatory bowel disease, and sarcoidosis. However, its role in modulating disease pathogenesis is still unknown. In this study, we found that IL-27 production is induced by TNF-α in human macrophages (MΦ) and investigated the effects of IL-27 on the responses of primary human MΦ to the endogenous inflammatory cytokines TNF-α and IL-1. In striking contrast to IL-27-mediated augmentation of TLR-induced cytokine production, we found that IL-27 suppressed MΦ responses to TNF-α and IL-1β, thus identifying an anti-inflammatory function of IL-27. IL-27 blocked the proximal steps of TNF-α signaling by downregulating cell-surface expression of the signaling receptors p55 and p75. The mechanism of inhibition of IL-1 signaling was downregulation of the ligand-binding IL-1RI concomitant with increased expression of the receptor antagonist IL-1Ra and the decoy receptor IL-1RII. These findings provide a mechanism for suppressive effects of IL-27 on innate immune cells and suggest that IL-27 regulates inflammation by limiting activation of MΦ by inflammatory cytokines while preserving initial steps in host defense by augmenting responses to microbial products.     

IL-1R2 expression in human gastric cancer and its clinical significance

Background: Interleukin-1 receptor type II (IL-1R2), also known as CD121b, is a member of the IL-1 receptor family. IL-1R2 acts as negative regulator of the IL-1 system, modulating IL-1 availability for the signaling receptor. IL-1R2 is abnormally expressed in many human inflammatory diseases and cancers, and has important clinical significance. The present study was designed to investigate IL-1R2 expression in human gastric cancer (GC) tissues and the associated clinical implications. Methods: Immunohistochemistry was used to identify the clinical significance and prognostic value of IL-1R2 expression in GC tissues. We investigated IL-1R2 expression in GC tissues, cells, and serum using real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) assays. Results: IL-1R2 was highly expressed in GC tissues, and the overall survival in patients with advanced GC and high IL-1R2 expression was significantly poorer than that in patients with advanced GC and low IL-1R2 expression. Moreover, IL-1R2 mRNA levels in GC tissues and most GC cells were higher than those in para-cancer tissues and GES1 human gastric mucosal epithelial cells. The level of plasma-soluble IL-1R2 in GC patients was higher than that of the healthy control group. Conclusion: Increased IL-1R2 levels are involved in the initiation and progression of human GC, and IL-1R2 might be employed to develop immunotherapeutic approaches targeting GC.     

Th17 can regulate silica‐induced lung inflammation through an IL‐1β‐dependent mechanism

Silicosis is an occupational lung disease caused by the inhalation of silica dust and characterized by lung inflammation and fibrosis. Interleukin (IL)‐1β is induced by silica and functions as the key pro‐inflammatory cytokine in this process. The Th17 response, which is induced by IL‐1β, has been reported very important in chronic human lung inflammatory diseases. To elucidate the underlying mechanisms of IL‐1β and IL‐17 in silicosis, we used anakinra and an anti‐IL‐17 monoclonal antibody (mAb) to block the receptor of IL‐1β (IL‐RI) and IL‐17, respectively, in a mouse model of silicosis. We observed increased IL‐1β expression and an enhanced Th17 response after silica instillation. Treatment with an IL‐1 type I receptor (IL‐1RI) antagonist anakinra substantially decreased silica‐induced lung inflammation and the Th17 response. Lung inflammation and the accumulation of inflammatory cells were attenuated in the IL‐17‐neutralized silicosis group. IL‐17 may promote lung inflammation by modulating the differentiation of Th1 and regulatory T cells (Tregs) and by regulating the production of IL‐22 and IL‐1β during the lung inflammation of silicosis. Silica may induce IL‐1β production from alveolar macrophages and promote inflammation by initiating a Th17 response via an IL‐1β/IL‐1RI‐dependent mechanism. The Th17 response could induce lung inflammation during the pathogenesis of silicosis by regulating the homoeostasis of the Th immune responses and affecting the production of IL‐22 and IL‐1β. This study describes a potentially important inflammatory mechanism of silicosis that may bring about novel therapies for this inflammatory and fibrotic disease.     

Enhanced CCL26 production by IL-4 through IFN-gamma-induced upregulation of type 1 IL-4 receptor in keratinocytes

A Th2 cytokine, IL-4, induces various chemokines from epidermal keratinocytes which play crucial roles in the pathogenesis of skin disorders such as atopic dermatitis. In contrast, the role of IFN-γ, a Th1 cytokine, on eosinophilic skin inflammation is unclear. This study investigated the effects of IFN-γ on IL-4-induced production of eotaxin-3/CCL26, a potent chemoattractant to eosinophils, in normal human epidermal keratinocytes (NHEK). When the cells were stimulated with IL-4 and IFN-γ simultaneously, IL-4-induced CCL26 production was attenuated. In contrast, prior stimulation with IFN-γ enhanced IL-4-induced CCL26 production. NHEK constitutively expressed type 1 IL-4 receptor, and expression at the cell surface was upregulated by stimulation with IFN-γ. This upregulation resulted in an enhanced IL-4-mediated cellular signal. These results indicate that IFN-γ has opposite effects on IL-4-induced CCL26 production in NHEK depending on the time of exposure. Thus, changes in IL-4R expression by IFN-γ might modulate eosinophilic skin inflammation.     

Mutations in IL36RN/IL1F5 are associated with the severe episodic inflammatory skin disease known as generalized pustular psoriasis

Generalized pustular psoriasis (GPP) is a rare and yet potentially lethal clinical variant of psoriasis, characterized by the formation of sterile cutaneous pustules, neutrophilia, fever and features of systemic inflammation. We sequenced the exomes of five unrelated individuals diagnosed with GPP. Nonsynonymous, splice-site, insertion, and deletion variants with an estimated population frequency of <0.01 were considered as candidate pathogenic mutations. A homozygous c.338C>T (p.Ser113Leu) missense substitution of IL36RN was identified in two individuals, with a third subject found to be a compound heterozygote for c.338C>T (p.Ser113Leu) and a c.142C>T (p.Arg48Trp) missense substitution. IL36RN (previously known as IL1F5) encodes an IL-1 family receptor antagonist, which opposes the activity of the IL-36A and IL-36G innate cytokines. Homology searches revealed that GPP mutations alter evolutionarily conserved residues. Homozygosity for the c.338C>T (p.Ser113Leu) variant is associated with an elevated proinflammatory response following ex vivo stimulation with IL36A. These findings suggest loss of function of IL36RN as the genetic basis of GPP and implicate innate immune dysregulation in this severe episodic inflammatory disease, thereby highlighting IL-1 signaling as a potential target for therapeutic intervention.     

Important roles of CD32 in promoting suppression of IL-4 induced immune responses by a novel anti-IL-4Rα therapeutic antibody

ABSTRACT

Asthma is characterized by airway hyperresponsiveness and inflammation, as well as underlying structural changes to the airways. Interleukin-4 (IL-4) is a key T-helper type 2 (Th2) cytokine that plays important roles in the pathogenesis of atopic and eosinophilic asthma. We developed a novel humanized anti-IL-4Rα antibody that can potently inhibit IL-4/IL-13-mediated TF-1 cell proliferation. Using monocytes isolated from human peripheral blood mononuclear cells (PBMCs), we revealed a critical role of CD32 in modulating the immune responses of monocytes in response to blockade of IL-4Rα signaling pathway. We, therefore, devised a new strategy to increase the efficacy of the anti-IL-4Rα monoclonal antibody for the treatment of asthma and other atopic diseases by co-engaging CD32 and IL-4Rα on monocytic cells by choosing IgG classes or Fc mutations with higher affinities for CD32. The antibody with selectively enhanced affinity for CD32A displayed superior suppression of IL-4-induced monocytes’ activities, including the down-regulation of CD23 expression. Intriguingly, further analysis demonstrated that both CD32A and CD32B contributed to the enhancement of antibody-mediated suppression of CD23 expression from monocytes in response to blockade of IL-4Rα signaling. Furthermore, inhibition of IgE secretion from human PBMC by the antibody variants further suggests that the complex allergic inflammation mediated by IL-4/IL-4Rα signaling might result from a global network where multiple cell types that express multiple FcγRs are all involved, of which CD32, especially CD32A, is a key mediator. In this respect, our study provides new insights into designing therapeutic antibodies for targeting Th2 cytokine-mediated allergic pathogenesis.     

Acid-dependent Interleukin-1 (IL-1) Cleavage Limits Available Pro-IL-1β for Caspase-1 Cleavage

Noncommunicable diseases such as cardiovascular disease (stroke and heart attack), cancer, chronic respiratory disease, and diabetes are a leading cause of death and disability worldwide and are worsened by inflammation. IL-1 is a driver of inflammation and implicated in many noncommunicable diseases. Acidosis is also a key feature of the inflammatory microenvironment; therefore it is vital to explore IL-1 signaling under acidic conditions. A HEK-IL-1 reporter assay and brain endothelial cell line were used to explore activity of mature IL-1α and IL-1β at pH 7.4 and pH 6.2, an acidic pH that can be reached under inflammatory or ischemic conditions, alongside cathepsin D-cleaved 20-kDa IL-1β produced under acidic conditions. We report that mature IL-1 signaling at IL-1 receptor type 1 (IL-1R1) is maintained at pH 6.2, but the activity of the decoy receptor, IL-1R2, is reduced. Additionally, cathepsin D-cleaved 20-kDa IL-1β was minimally active at IL-1R1 and was not further cleaved to highly active 17-kDa IL-1β. Therefore formation of the 20-kDa form of IL-1β may prevent the generation of mature bioactive IL-1β and thus may limit inflammation.     

Oncostatin M (OSM) primes IL-13- and IL-4-induced eotaxin responses in fibroblasts: Regulation of the type-II IL-4 receptor chains IL-4Rα and IL-13Rα1

Oncostatin M (OSM), a pleiotropic cytokine and a member of the gp130/IL-6 cytokine family, has been implicated in regulation of various chronic inflammatory processes. Previous work has shown that OSM induces eosinophil accumulation in mouse lungs in vivo and stimulates the eosinophil-selective chemokine eotaxin-1 synergistically with IL-4 in vitro. To examine the role of receptor regulation by OSM in synergistic eotaxin-1 responses, we here examine the modulation of the type-II IL-4 receptor (IL-4Rα and IL-13Rα1) by OSM and other gp130/IL-6 cytokine family members using NIH3T3 fibroblasts and primary mouse lung fibroblasts. We first show that OSM with either IL-13 or IL-4 synergistically induces eotaxin-1 expression in a dose-dependent fashion. Analysis of IL-4Rα expression at the protein (Western blot and FACS) and RNA (TAQMAN) levels showed that OSM markedly elevates expression by 3 h. OSM enhanced IL-13Rα1 mRNA and induced a smaller but detectable increase in total IL-13Rα1 protein. Priming fibroblasts with OSM for 6 h markedly enhanced subsequent IL-13 and IL-4-induced eotaxin-1 responses and STAT6 tyrosine-641 phosphorylation. Regulation of IL-4Rα by OSM was sensitive to inhibition of the PI3′K pathway by LY294002. These studies provide novel mechanistic insights in OSM role in regulation of synergistic eotaxin-1 responses and IL-4Rα expression in fibroblasts.     

Role of interleukin (IL)-1 type 1 receptor in mycobacterial infection.

It is important to gain a better understanding of IL-1-mediated signaling events in mycobacterial infection. In order to clarify the role of IL-1 receptor type 1 (IL-1 R1) in IL-1 R1, knockout (KO) mice were infected with either Mycobacterium tuberculosis H37Rv or Kurono strain by the respiratory route, and their ability to control mycobacterial growth, pulmonary granuloma formation, and cytokine mRNA expression was investigated. IL-1 R1 KO mice developed significantly larger (P< 0.01) granulomatous lesions with neutrophil infiltration in their lungs than wild-type mice did after infection with the M. tuberculosis Kurono strain. The number of mycobacterial colonies in lungs and spleen increased from five weeks post-infection. Interferon-y production by spleen cells was low in IL-1 R1 KO mice. It is concluded that the IL-1 R1 is essential for IL-1-mediated signaling events in mycobacterial infection.     

A complicated liaison: IL-33 and IL-33R in arthritis pathogenesis

Interruption of cytokine signaling, by targeting either the cytokine itself or its cellular receptor, is a mainstay in the therapy for patients with rheumatic diseases. Interleukin (IL)-33, a member of the IL-1 cytokine family, has emerged as an important mediator of inflammatory responses. In a side-by-side examination of IL-33-deficient and IL-33 receptor (IL-33R)-deficient mice in the K/BxN serum transfer model, arthritis was ameliorated in the IL-33R knockout (KO) mice but not in the IL-33 KO mice. These findings complement previous knowledge on IL-33R signaling, demonstrating that the IL-33R cross-activates other signaling pathways in addition to IL-33-mediated signals. The results reported by Martin and colleagues in a previous issue of Arthritis Research & Therapy underline the clinical relevance of IL-33R cross-signaling and further illustrate that targeting a cytokine receptor (IL-33R) can have completely different clinical outcomes than targeting the respective cytokine.     

Altered expression of inflammatory cytokine receptors in response to LPS challenge through interaction between intestinal epithelial cells and lymphocytes of Peyer's patch

The intense innate immunological activities occurring at the enteric mucosal surface involve interactions between intestinal epithelial cells and immune cells. Our previous studies have indicated that Peyer's patch lymphocytes may modulate intestinal epithelial barrier and ion transport function in homeostasis and host defense via cell-cell contact as well as cytokine signaling. The present study was undertaken using the established co-culture system of Caco-2 epithelial cells with lymphocytes of Peyer's patch to investigate the expression of IL-8 and IL-6 cytokines and cytokine receptors in the co-culture system after challenge with Shigella F2a-12 lipopolysaccharide (LPS). The human colonic epithelial cell line Caco-2 was co-cultured with freshly isolated lymphocytes from the murine Peyer's patch either in the mixed or separated (isolated but permeable compartments) co-culture configuration, and was challenged with Shigella F2a-12 LPS for 8 h. The level of mRNA expressions of human interleukin-8 (hIL-8), human interleukin-8 receptor (hIL-8R), mouse interleukin-8 receptor (mIL-8R), mouse interleukin-6 (mIL-6), mouse interleukin-6 receptor (mIL-6R) and human interleukin-6 receptor (hIL-6R) was examined by semi-quantitative PCR. In both co-culture groups, hIL-8 expression of Caco-2 cells was decreased, and hIL-8R expression was increased compared to the Caco-2 alone group. Upon LPS challenge, hIL-8 expression from the Caco-2 cells of both co-culture groups was higher than in the Caco-2 control group. The increased hIL-8 expression of Caco-2 cells in the separated co-culture group is correlated with a decreased hIL-8R expression and an increased mIL-8R expression. In the mixed co-culture group, the increased expression of hIL-8 was associated with the upregulated hIL-8R expression on Caco-2 cells and downregulated mIL-8R on murine Peyer's patch lymphocytes (PPL). mIL-6 expression from mouse PPL was also upregulated by LPS in mixed co-culture. However, upon the treatment with LPS, hIL-6R expression of Caco-2 cells was decreased in the mixed co-culture, but increased in separated co-culture. The data suggest that release of hIL-8 from epithelial cells may act on lymphocytes through a paracrine pathway, but it may also act on the epithelial cells themselves via an autocrine pathway. The data also suggest that the release of mIL-6 from Peyer's patch lymphocytes affects epithelial cells in a paracrine fashion.