Generation and Characterization of Inducible Lung and Skin-Specific IL-22 Transgenic Mice.

IL-22 is an IL-10 family cytokine that is increased in asthma and atopic dermatitis (AD). However, the specific role of IL-22 in the pathogenesis of allergic lung inflammation and AD in vivo has yet to be elucidated. We aimed to develop mouse models of allergic diseases in the lung and skin with inducible and tissue-specific expression of IL-22, using a tetracycline (Tet)-controlled system. In this chapter, we describe a series of protocols we have developed to generate a construct that contains the TRE-Tight promoter and mouse IL-22 cDNA based on this system. Furthermore, we describe how to generate TRE-Tight-IL-22 mice through pronuclear microinjection. In our approach, two Tet-on (CC10-rtTA or SPC-rtTA) and a Tet-off (K5-tTA) transgenic mouse lines are selected to crossbreed with TRE-Tight-IL-22 mice to generate inducible tissue-specific transgenic lines. The transgenic strains, CC10-rtTA/TRE-Tight-IL-22 (CC10-rtTA-IL-22) or SPC-rtTA/TRE-Tight-IL-22 (SPC-rtTA-IL-22) mice, do not produce detectable levels of IL-22 in their bronchoalveolar lavage (BAL) samples in the absence of doxycycline (Dox). However, oral Dox treatment of these mice induces IL-22 expression in the BAL, and the airway and lung epithelial cells. For K5-tTA/TRE-Tight-IL-22 (K5-tTA-IL-22) mice, to avoid potential IL-22 toxicity to mouse embryos, Dox is given starting at the time of breeding to suppress tTA and to keep the IL-22 transgene off until the K5-tTA-IL-22 mice are 6 weeks old. Experiments are then initiated by withdrawing Dox from the drinking water. In all cases, IL-22 protein can be detected by immunohistochemistry in the skin of Tg(+) animals, but not in the skin of Tg(-) animals. Utilizing transgenic technology based on the Tetracycline-controlled system, we have established inducible transgenic mouse models in which cytokine IL-22 can be expressed specifically in the lung or skin. These models are valuable for studies in vivo in a broad range of diseases involving IL-22 and will provide a new platform for research and for seeking novel therapeutics in the fields of inflammation, asthma, and allergic dermatitis.

IL-35 and RANKL Synergistically Induce Osteoclastogenesis in RAW264 Mouse Monocytic Cells.

Interleukin (IL)-35 is an immunosuppressive cytokine mainly produced by regulatory T cells. IL-35 mediates immunological functions by suppressing the inflammatory immune response. However, the role of IL-35 in bone-destructive diseases remains unclear, especially in terms of osteoclastogenesis. Therefore, the current study investigated the synergistic effect of IL-35 on osteoclastogenesis that is involved the pathogeneses of periodontitis and rheumatoid arthritis. Osteoclastic differentiation and osteoclastogenesis of RAW264 (RAW) cells induced by receptor activator of nuclear factor (NF)-κB ligand (RANKL) and IL-35 were evaluated by tartrate-resistant acid phosphate staining, hydroxyapatite resorption assays, and quantitative polymerase chain reaction. The effect of IL-35 on RANKL-stimulated signaling pathways was assessed by Western blot analysis. Costimulation of RAW cells by RANKL and IL-35 induced osteoclastogenesis significantly compared with stimulation by RANKL alone. Phosphorylations of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase tended to be increased by RANKL and IL-35 compared with RANKL or IL-35 alone. Additionally, the osteoclastogenesis induced by RANKL and IL-35 was suppressed by inhibition of ERK. In this study, IL-35 and RANKL induced osteoclastogenesis synergistically. Previous reports have shown that IL-35 suppresses the differentiation of osteoclasts. Therefore, IL-35 might play dual roles of destruction and protection in osteoclastogenesis.

An Open-Label, Dose-Escalation Study to Assess the Safety and Efficacy of IL-22 Agonist F-652 in Patients With Alcohol-associated Hepatitis.

BACKGROUND AND AIMS: Interleukin-22 has beneficial effects on inflammation and impaired hepatic regeneration that characterize alcohol-associated hepatitis (AH). F-652 is a recombinant fusion protein of human interleukin-22 and immunoglobulin G2 fragment crystallizable. This study aims to assess the safety and efficacy signals of F-652 in patients with moderate and severe AH. APPROACH AND RESULTS: A phase-2 dose-escalating study was carried out. F-652 (10 µg/kg, 30 µg/kg, or 45 µg/kg) administered on days 1 and 7 was tested in 3 patients each with moderate (Model for End-Stage Liver Disease [MELD] scores: 11-20) and severe AH (MELD scores: 21-28). Safety was defined by absence of serious adverse events and efficacy was assessed by Lille score, changes in MELD score, and serum bilirubin and aminotransferases at days 28 and 42. Three independent propensity-matched comparator patient cohorts were used. Plasma extracellular vesicles and multiplex serum cytokines were measured to assess inflammation and hepatic regeneration. Eighteen patients (9 moderate and 9 severe AH) were enrolled, 66% were male, and the mean age was 48 years. The half-life of F-652 following the first dose was 61-85 hours. There were no serious adverse events leading to discontinuation. The MELD score and serum aminotransferases decreased significantly at days 28 and 42 from baseline (P < 0.05). Day-7 Lille score was 0.45 or less in 83% patients as compared with 6%, 12%, and 56% among the comparator cohorts. Extracellular vesicle counts decreased significantly at day 28 (P < 0.013). Cytokine inflammatory markers were down-regulated, and regeneration markers were up-regulated at days 28 and 42. CONCLUSIONS: F-652 is safe in doses up to 45 µg/kg and associated with a high rate of improvement as determined by Lille and MELD scores, reductions in markers of inflammation and increases in markers of hepatic regeneration. This study supports the need for randomized placebo-controlled trials to test the efficacy of F-652 in AH.

Effect of folate supplementation on immunological and autophagy markers in experimental nonalcoholic fatty liver disease.

BACKGROUND AND AIMS: Chronic hepatic inflammation is an important pathogenic mediator of nonalcoholic fatty liver disease (NAFLD) that contributes to disease severity. It is commonly suggested that autophagy dysfunction may be an underlying cause of nonalcoholic fatty liver disease. However, the exact role of autophagy in lipid metabolism remains controversial. There has been a growing interest in the role of folate supplementation for the treatment and/or prevention of NAFLD. We aimed in this study to investigate the effects of different doses of folate supplementation on several immune markers and autophagy trying to explore the complex role of IL-22 and autophagy in NAFLD. METHODS: Fifty Wistar rats were randomly separated into experimental (n = 40) and control groups (n = 10), which were fed for eight weeks with a high-fat diet (HFD) containing 40% fats or a standard diet, respectively. The experimental group was further subdivided into four subgroups where the first subgroup was left untreated while the other three were treated with different doses of folate (50, 100, and 150 µg/kg of body weight, respectively). At the end of the experimental period, animals from each group were sacrificed for blood and tissue analyses. RESULTS: NAFLD rats showed decreased IL-22 serum levels and increased LC3B expression as compared to controls. Folate treatment was significantly associated with improvement in disease parameters, reduced presence of the pro-inflammatory cytokines TNF-α and CXCL8 and LC3B expression, and increased IL-22 levels in a dose-dependent manner. CONCLUSION: These results highlight the capacity of folate to modulate the production of several pro-inflammatory cytokines and autophagy thereby having a favorable impact disease progression.

Glycyrrhizin alleviates Con A-induced hepatitis by differentially regulating the production of IL-17 and IL-25.

Glycyrrhizin, a triterpenoid compound, has been reported to be an anti-inflammatory agent for the treatment of a variety of inflammatory diseases including hepatitis. However, the mechanism by which glycyrrhizin inhibits inflammation is unclear. Using a Con A-induced hepatitis model in mice, we found that administration of glycyrrhizin ameliorates Con A-induced liver injury, which manifests as reduction in the production of inflammatory cytokines IFN-γ, IL-6 and IL-17, as well as serum alanine aminotransferase (ALT). Blockade of IL-17 dramatically mitigates liver injury resulting from Con A challenge. Interestingly, at both the mRNA and protein levels, the endogenous alarmin inflammatory molecule high-mobility group box 1 (HMGB1) is significantly decreased in mice injected with glycyrrhizin combined with Con A compared to those injected with Con A alone. In contrast, the administration of glycyrrhizin with Con A challenge up-regulates the production of IL-25. Furthermore, an increase in the proportion of protective lymphocyte subset, Gr-1+ CD11b+ (Myeloid-Derived Suppressor Cell, MDSCs), could be induced by increased IL-25 to restrain immune cell activation and favor the resolution of detrimental immune reactions caused by Con A challenge. The results indicate that glycyrrhizin plays a protective role in Con A-induced hepatitis. This protective role is particularly associated with reducing the production of IL-17 and enhancing the expression of IL-25. The present study may provide a new strategy for the treatment of acute hepatitis in the clinical setting.

Production of IL-35 by Bregs is mediated through binding of BATF-IRF-4-IRF-8 complex to il12a and ebi3 promoter elements.

IL-10 and IL-35 suppress excessive immune responses and therapeutic strategies are being developed to increase their levels in autoimmune diseases. In this study, we sought to identify major cell types that produce both cytokines in-vivo and to characterize mechanisms that regulate their production. Experimental autoimmune uveitis (EAU) is a CNS autoimmune disease that serves as model of human uveitis. We induced EAU in C57BL/6J mice and investigated whether T cells, B lymphocytes, or myeloid cells are the major producers of IL-10 or IL-35 in blood, lymph nodes (LNs), spleen, and at the site of ocular inflammation, the neuroretina. Analysis of these tissues identified B cells as the major producers of IL-10 and IL-35 in-vivo. Compared to regulatory T cells (Tregs), IL-10- or IL-35-producing regulatory B cells (Bregs) are substantially expanded in blood, LNs, spleen, and retina of mice with EAU. We performed EMSA and chromatin immunoprecipitation (ChIP) assays on activated B cells stimulated with IL-35 or TLR agonists. We found that BATF, IFN regulatory factor (IRF)-4, and IRF-8 transcription factors were recruited and bound to AP1-IRF-composite elements (AICEs) of il12a, ebi3, and/or il10 loci, suggesting their involvement in regulating IL-10 and IL-35 transcriptional programs of B cells. Showing that B cells are major source of IL-10 and IL-35 in-vivo and identifying transcription factors that contribute to IL-10 and IL-35 expression in the activated B-cell, suggest that the BATF/IRF-4/IRF-8 axis can be exploited therapeutically to regulate physiological levels of IL-10/IL-35-Bregs and that adoptive transfer of autologous Bregs might be an effective therapy for autoimmune and neurodegenerative diseases.

The Role of Interleukin-19 in Contact Hypersensitivity.

Interleukin (IL)-19 is a member of the IL-10 family of interleukins and is an immuno-modulatory cytokine produced by the main macrophages. The gastrointestinal tissues of IL-19 knockout mice show exacerbated experimental colitis mediated by the innate immune system and T cells. There is an increasing focus on the interaction and relationship of IL-19 with the function of T cells. Contact hypersensitivity (CHS) is T cell-mediated cutaneous inflammation. Therefore, we asked whether IL-19 causes CHS. We investigated the immunological role of IL-19 in CHS induced by 1-fluoro-2,4-dinitrofluorobenzene as a hapten. IL-19 was highly expressed in skin exposed to the hapten, and ear swelling was increased in IL-19 knockout mice. The exacerbation of the CHS response in IL-19 knockout mice correlated with increased levels of IL-17 and IL-6, but no alterations were noted in the production of interferon (IFN)γ and IL-4 in the T cells of the lymph nodes. In addition to the effect on T cell response, IL-19 knockout mice increased production of inflammatory cytokines. These results show that IL-19 suppressed hapten-dependent skin inflammation in the elicitation phase of CHS.

Immunostimulatory Defective Viral Genomes from Respiratory Syncytial Virus Promote a Strong Innate Antiviral Response during Infection in Mice and Humans.

Human respiratory syncytial virus (RSV) is a major cause of severe respiratory illness in children and susceptible adults. RSV blocks the development of the innate antiviral immune response and can grow to high titers in the respiratory tract. Here we demonstrate that immunostimulatory defective viral genomes (iDVGs) that are naturally generated during RSV replication are strong inducers of the innate antiviral response to RSV in mice and humans. In mice, RSV iDVGs stimulated the expression of antiviral genes, restricted viral replication, and prevented weight loss and lung inflammation. In human cells, the antiviral response to RSV iDVGs was dominated by the expression of IFN-λ1 over IFN-ß and was driven by rapid intranuclear accumulation of the transcription factor IRF1. RSV iDVGs were detected in respiratory secretions of hospitalized patients, and their amount positively correlated with the level of expression of antiviral genes in the samples. Infection of explanted human lung tissue from different donors revealed that most humans can respond to RSV iDVGs and that the rate of accumulation of iDVGs during infection directly correlates with the quality of the antiviral response. Taken together, our data establish iDVGs as primary triggers of robust antiviral responses to RSV and provide the first evidence for an important biological role for naturally occurring iDVGs during a paramyxovirus infection in humans.

The interleukin (IL)-1 cytokine family--Balance between agonists and antagonists in inflammatory diseases.

The interleukin (IL)-1 family of cytokines comprises 11 members, including 7 pro-inflammatory agonists (IL-1α, IL-1ß, IL-18, IL-33, IL-36α, IL-36ß, IL-36γ) and 4 defined or putative antagonists (IL-1R antagonist (IL-1Ra), IL-36Ra, IL-37, and IL-38) exerting anti-inflammatory activities. Except for IL-1Ra, IL-1 cytokines do not possess a leader sequence and are secreted via an unconventional pathway. In addition, IL-1ß and IL-18 are produced as biologically inert pro-peptides that require cleavage by caspase-1 in their N-terminal region to generate active proteins. N-terminal processing is also required for full activity of IL-36 cytokines. The IL-1 receptor (IL-1R) family comprises 10 members and includes cytokine-specific receptors, co-receptors and inhibitory receptors. The signaling IL-1Rs share a common structure with three extracellular immunoglobulin (Ig) domains and an intracellular Toll-like/IL-1R (TIR) domain. IL-1 cytokines bind to their specific receptor, which leads to the recruitment of a co-receptor and intracellular signaling. IL-1 cytokines induce potent inflammatory responses and their activity is tightly controlled at the level of production, protein processing and maturation, receptor binding and post-receptor signaling by naturally occurring inhibitors. Some of these inhibitors are IL-1 family antagonists, while others are IL-1R family members acting as membrane-bound or soluble decoy receptors. An imbalance between agonist and antagonist levels can lead to exaggerated inflammatory responses. Several genetic modifications or mutations associated with dysregulated IL-1 activity and autoinflammatory disorders were identified in mouse models and in patients. These findings paved the road to the successful use of IL-1 inhibitors in diseases that were previously considered as untreatable.

IL-21 enhances the activity of the TLR-MyD88-STAT3 pathway but not the classical TLR-MyD88-NF-κB pathway in human B cells to boost antibody production.

Both IL-21 and TLR agonists are important regulators of B cell responses, and the combination of IL-21 and TLR stimulation results in increased Ab production. However, it is not clear yet how IL-21 interacts with TLR signaling in B cells. In this study, we show that IL-21 enhances TLR-induced IgG production, whereas it has no effect on TLR-induced IL-6 production by human B cell cultures. These observations are explained by the finding that IL-21 augments TLR-induced IgG production via the TLR-MyD88-STAT3 pathway but not the classical TLR-MyD88-NF-κB pathway. We further demonstrate that stimulation of human B cells with IL-21 and TLR7/8 or TLR9 agonists increases the phosphorylation of STAT3, whereas the activation of NF-κB is not affected. Interestingly, like IL-21, IL-10 in combination with TLR signaling also enhances phosphorylation of STAT3, resulting in an increase of IgG production. Hence, IL-21 and IL-10 increase the activity of the TLR-MyD88-STAT3 pathway in human B cells via enhancing the phosphorylation of STAT3 for Ab production.

Targeting IL-23 and Th17-cytokines in inflammatory bowel diseases.

Over the last 15 years, the use of various biological therapies has largely improved the way we manage patients with Inflammatory Bowel Diseases (IBDs). Blockade of cytokine synthesis and/or activity is at the forefront of this new era with the success of inhibitors of tumor necrosis factor (TNF)-α. These therapies are however not effective in all IBD patients and efficacy may wane. Moreover, patients treated with anti-TNF-α antibodies can develop severe side-effects and new immune-mediated diseases. Therefore, a new challenge is to elucidate new inflammatory networks in the IBD tissue and develop novel anti-cytokine compounds, which may act in patients who are resistant to or cannot receive anti-TNF-α therapies. In this article we review the available data supporting the pathogenic role of IL-23 and Th17-related cytokines in IBD, and discuss whether and how compounds that control the activity of these cytokines may enter into the therapeutic armamentarium of IBD.

Recomendaciones para el uso de agentes biológicos en psoriasis / Recommendations for the use of biological agents in psoriasis

La psoriasis es una enfermedad crónica que afecta al 1%-2% de la población, produce importante deterioro de la calidad de vida y puede asociarse a complicaciones metabólicas y articulares severas. Existen múltiples agentes sistémicos para el manejo de los casos moderado-severos; sin embargo, la mayoría no puede utilizarse de manera continua o prolongada y los estudios han demostrado que un alto porcentaje de pacientes está disconforme con sus tratamientos. Los agentes biológicos representan una poderosa herramienta en el tratamiento de la psoriasis; no obstante, los riesgos asociados y sus costos hacen necesario restringir su uso. Debido a estas consideraciones, diferentes sociedades dermatológicas en el mundo han desarrollado guías clínicas de consenso para el manejo de estos agentes en psoriasis. El presente artículo resume las recomendaciones de las sociedades Alemana (2007), Americana (2008), Española (2009) y Británica (2009) de Dermatología y los principales ensayos clínicos de cada uno de los agentes biológicos disponibles para el tratamiento de la psoriasis, y pretende entregar algunas directrices para la utilización de estos agentes en el medio nacional.

Psoriasis is a chronic disease that affects 1%-2% of the population, causes serious deterioration in the quality of life and may be associated with severe metabolic and joint complications. There are numerous systemic agents for the management of moderate-severe cases, however, most of them cannot be used in a continuous or prolonged way, and studies have shown a high percentage of dissatisfaction with those treatments. Biologics represent a powerful tool in the treatment of psoriasis, however, associated risks and costs make it necessary to restrict their use. Because of this, different dermatological societies around the world have developed clinical guidelines for the management of these agents in psoriasis. This article summarizes the recommendations of the German (2007), American (2008), Spanish (2009) and British (2009) Dermatology Societies, as well as the main clinical trials of biologics for the treatment of Psoriasis, and attempts to provide some guidelines for the use of these agents in our country.

A proinflammatory cytokine interleukin-32beta promotes the production of an anti-inflammatory cytokine interleukin-10.

A new proinflammatory cytokine interleukin-32 (IL-32) has six isoforms. Although IL-32 can be detected in sera from patients suffering from Crohn's disease and rheumatoid arthritis, it is unclear which isoforms are involved. To this end, we investigated the functions of the most abundant IL-32beta by generating K562-IL-32beta stable cell lines. This report confirms, using IL-32 small interfering RNA, that IL-32beta induces an anti-inflammatory cytokine IL-10 in K562-IL-32beta cells and U937 promonocytic cells, which express endogenous IL-32beta upon phorbol 12-myristate 13-acetate (PMA) treatment, and monocyte-derived dendritic cells (DC) upon lipopolysaccharide (LPS) treatment. Interleukin-32beta was induced in monocyte-derived macrophages by LPS and in monocyte-derived DC by LPS, poly(I:C), or anti-CD40 antibody, but was not induced by PMA. We showed that IL-32beta expression was increased in a time-dependent manner in monocyte-derived DC upon LPS treatment and peaked at 24 hr. Production of IL-10 was exactly coincident with IL-32beta expression, but IL-1beta and tumour necrosis factor-alpha production peaked at 6 hr after LPS treatment, then steeply declined. Interleukin-12 p40 was induced at 9 hr and gradually increased until 48 hr, at which time IL-32beta and IL-10 were no longer increased. Knock-down of IL-32beta by IL-32 small interfering RNA led to the decrease of IL-10, but the increase of IL-12 in monocyte-derived DC, which means that IL-32beta promotes IL-10 production, but limits IL-12 production. We also showed that IL-10 neutralization increases IL-12, IL-1beta and tumour necrosis factor-alpha production, which implies that IL-10 suppresses such proinflammatory cytokines. Taken together, our results suggest that IL-32beta upregulates the production of an anti-inflammatory cytokine IL-10, and then IL-10 suppresses proinflammatory cytokines.

The combination of IL-21 and IFN-alpha boosts STAT3 activation, cytotoxicity and experimental tumor therapy.

For decades cytokines such as type I interferons and IL-2 have been used in immunotherapy against cancer, viral hepatitis, and autoimmune diseases such as multiple sclerosis. However, the therapeutic use of cytokines has been hampered by their pleiotropic effects on target-cells. Thus, cytokines such as IFN-alpha and IL-2 have multiple and severe side effects. Accordingly, they are generally used at sub-optimal doses, which limit their clinical efficacy. Here we hypothesized that a combination of IFN-alpha and IL-21, a novel cytokine of the IL-2 family with anti-cancer effects, will increase the anti-cancer efficacy at sub-optimal cytokine doses. We show that the combined stimulation of target-cells with IFN-alpha and IL-21 triggers an increased STAT3 activation whereas the activation of other STATs including STAT1/2 is unaffected. In parallel, the combined stimulation with IFN-alpha and IL-21 triggers a selective increase in MHC class I expression and NK- and CD8(+) T-cell-mediated cytotoxicity. In an experimental in vivo model of renal carcinoma, the combined treatment of IFN-alpha and IL-21 also produces a significant anti-cancer effect as judged by an inhibition of tumor growth and an increased survival. Taken together our data show that the combined use of IFN-alpha and IL-21 boosts STAT3 signaling, cytotoxicity, and anti-tumor efficacy, suggesting that a combinatorial therapeutic use of these cytokines may benefit cancer patients.

The IL-33/ST2 pathway: therapeutic target and novel biomarker.

For many years, the interleukin-1 receptor family member ST2 was an orphan receptor that was studied in the context of inflammatory and autoimmune disease. However, in 2005, a new cytokine--interleukin-33 (IL-33)--was identified as a functional ligand for ST2. IL-33/ST2 signalling is involved in T-cell mediated immune responses, but more recently, an unanticipated role in cardiovascular disease has been demonstrated. IL-33/ST2 not only represents a promising cardiovascular biomarker but also a novel mechanism of intramyocardial fibroblast-cardiomyocyte communication that may prove to be a therapeutic target for the prevention of heart failure.

IL-21 and BAFF/BLyS synergize in stimulating plasma cell differentiation from a unique population of human splenic memory B cells.

Both constitutive Ig secretion by long-lived plasma cells (PC) and the recurrent differentiation of memory (mem) B cells into PC contribute to the maintenance of serologic mem. However, the relative contribution of each is unknown. In this study, we describe a novel population of human postswitched mem B cells that rapidly differentiate into PC and thus contribute to serologic mem. These IgG(+) B cells reside in the region of human spleen analogous to the murine marginal zone and have not previously been examined. These cells are highly responsive to IL-21 in the context of CD40 stimulation. Uniquely, IgG(+) marginal zone analog B cells are exquisitely sensitive to the combination of IL-21 and B cell-activating factor belonging to the TNF family (BAFF/BLyS) that synergize in the absence of further costimulation to induce up-regulation of B lymphocyte-induced maturation protein-1 and drive PC differentiation. Other cytokine combinations are not active in this regard. This is the first demonstration that this unique population of mem B cells can respond specifically and exclusively to IL-21 and BAFF/BLyS by differentiating into IgG-secreting PC, and thus contributing to serologic mem in an Ag-independent manner.

In vivo and in vitro roles of IL-21 in inflammation.

IL-21 is a cytokine known to mediate its biological action via the IL-21R, composed of a specific chain, IL-21Ralpha, and the common gamma-chain (CD132). Recent data suggest that IL-21 possesses proinflammatory properties. However, there is no clear evidence that IL-21 induces inflammation in vivo and, curiously, the interaction between IL-21 and neutrophils has never been investigated, despite the fact that these cells express CD132 and respond to other CD132-dependent cytokines involved in inflammatory disorders. Using the murine air pouch model, we found that IL-21 induced inflammation in vivo, based on recruitment of neutrophil and monocyte populations. In contrast to LPS, administration of IL-21 into the air pouch did not significantly increase the concentration of IL-6, CCL5, CCL3, and CXCL2. We demonstrated that HL-60 cells expressed IL-21Ralpha, which is down-regulated during their differentiation toward neutrophils, and that IL-21Ralpha is not detected in neutrophils. Concomitant with this, IL-21 induced Erk-1/2 phosphorylation in HL-60 cells, but not in neutrophils. To eliminate the possibility that IL-21 could activate neutrophils even in the absence of IL-21Ralpha, we demonstrated that IL-21 did not modulate several neutrophil functions. IL-21-induced Erk-1/2 phosphorylation was not associated with proliferation or differentiation of HL-60 toward neutrophils, monocytes, or macrophages. IL-21Ralpha was detected in human monocytes and monocyte-derived macrophages, but IL-21 increased CXCL8 production only in monocyte-derived macrophages. We conclude that IL-21 is a proinflammatory cytokine, but not a neutrophil agonist. We propose that IL-21 attracts neutrophils indirectly in vivo via a mechanism independent of IL-6, CCL3, CCL5, and CXCL2 production.