Intracellular Major Histocompatibility Complex Class II and C-X-C Motif Chemokine Ligand 10-Expressing Neutrophils Indicate the State of Anti-Tumor Activity Induced by Bacillus Calmette-Guérin.

(1) Background: Inflammatory responses induce the formation of both anti-tumor and pro-tumor neutrophils known as myeloid-derived suppressor cells (MDSCs). Intermittent intravesical infusion of Bacillus Calmette-Guérin (BCG) is an established cancer immunotherapy for non-muscle-invasive bladder cancer (NMIBC). However, the types of neutrophils induced via the inflammatory response to both tumor-bearing and BCG remain unclear. (2) Methods: We therefore analyzed neutrophil dynamics in the peripheral blood and urine of patients with NMIBC who received BCG therapy. Further, we analyzed the effects of BCG in a mouse intraperitoneal tumor model. (3) Results: BCG therapy induced the formation of CXCL10 and MHC class II-positive neutrophils in the urine of patients with NMIBC but did not induce MDSC formation. CXCL10- and MHC class II-expressing neutrophils were detected in peritoneal exudate cells formed after BCG administration. Partial neutrophil depletion using an anti-Ly6G antibody suppressed the upregulation of CXCL10 and MHC class II in neutrophils and reversed the anti-tumor activity of BCG in mouse models. (4) Conclusions: These results indicated that intracellular MHC class II- and CXCL10-expressing neutrophils indicate the state of anti-tumor activity induced via BCG. The status of neutrophils in mixed inflammation of immunosuppressive and anti-tumor responses may therefore be useful for evaluating immunological systemic conditions.

Initial Myeloid Cell Status Is Associated with Clinical Outcomes of Renal Cell Carcinoma.

The therapeutic outcome of immune checkpoint inhibition (ICI) can be improved through combination treatments with ICI therapy. Myeloid-derived suppressor cells (MDSCs) strongly suppress tumor immunity. MDSCs are a heterogeneous cell population, originating from the unusual differentiation of neutrophils/monocytes induced by environmental factors such as inflammation. The myeloid cell population consists of an indistinguishable mixture of various types of MDSCs and activated neutrophils/monocytes. In this study, we investigated whether the clinical outcomes of ICI therapy could be predicted by estimating the status of the myeloid cells, including MDSCs. Several MDSC indexes, such as glycosylphosphatidylinositol-anchored 80 kD protein (GPI-80), CD16, and latency-associated peptide-1 (LAP-1; transforming growth factor-ß1 precursor), were analyzed via flow cytometry using peripheral blood derived from patients with advanced renal cell carcinoma (n = 51) immediately before and during the therapy. Elevated CD16 and LAP-1 expressions after the first treatment were associated with a poor response to ICI therapy. Immediately before ICI therapy, GPI-80 expression in neutrophils was significantly higher in patients with a complete response than in those with disease progression. This is the first study to demonstrate a relationship between the status of the myeloid cells during the initial phase of ICI therapy and clinical outcomes.

Hemoglobin ß Expression Is Associated with Poor Prognosis in Clear Cell Renal Cell Carcinoma.

BACKGROUND: The regulation of the redox balance in the tumor microenvironment is thought to be an adaptive response of tumor cells to hypoxic environments. In recent years, it has been reported that the hemoglobin ß-chain (HBB), which is involved in scavenging reactive oxygen species (ROS), is expressed in several carcinomas. However, the relationship between HBB expression and the prognosis of renal cell carcinoma (RCC) remains unclear. METHODS: HBB expression was immunohistochemically analyzed in 203 nonmetastatic clear cell RCC (ccRCC) cases. Cell proliferation, invasion, and ROS production were measured in ccRCC cell lines treated with HBB-specific siRNA. RESULTS: The prognosis of HBB-positive patients was worse than that of HBB-negative patients. Cell proliferation and invasion were inhibited, and ROS production was increased by treatment with HBB-specific siRNA. Oxidative stress increased HBB expression in cells exposed to H2O2. CONCLUSIONS: HBB expression in ccRCC contributes to cancer cell proliferation by suppressing ROS production under hypoxic conditions. Taken together with clinical results and in vitro experiments, HBB expression may serve as a new prognostic biomarker for RCC in the future.

GPI-80 Augments NF-κB Activation in Tumor Cells.

Recent studies have discovered a relationship between glycosylphosphatidylinositol (GPI)-anchored protein 80 (GPI-80)/VNN2 (80 kDa GPI-anchored protein) and malignant tumors. GPI-80 is known to regulate neutrophil adhesion; however, the action of GPI-80 on tumors is still obscure. In this study, although the expression of GPI-80 mRNA was detectable in several tumor cell lines, the levels of GPI-80 protein were significantly lower than that in neutrophils. To clarify the function of GPI-80 in tumor cells, GPI-80-expressing cells and GPI-80/VNN2 gene-deleted cells were established using PC3 prostate cancer cells. In GPI-80-expressing cells, GPI-80 was mainly detected in vesicles. Furthermore, soluble GPI-80 in the conditioned medium was associated with the exosome marker CD63 and was also detected in the plasma obtained from prostate cancer patients. Unexpectedly, cell adhesion and migration of GPI-80-expressing PC3 cells were not modulated by anti-GPI-80 antibody treatment. However, similar to the GPI-80 family molecule, VNN1, the pantetheinase activity and oxidative state were augmented in GPI-80-expressing cells. GPI-80-expressing cells facilitated non-adhesive proliferation, slow cell proliferation, NF-κB activation and IL-1ß production. These phenomena are known to be induced by physiological elevation of the oxidative state. Thus, these observations indicated that GPI-80 affects various tumor responses related to oxidation.

Interleukin-21 in Viral Infections.

Interleukin (IL)-21 is a cytokine that affects the differentiation and function of lymphoid and myeloid cells and regulates both innate and adaptive immune responses. In addition to regulating the immune response to tumor and viral infections, IL-21 also has a profound effect on the development of autoimmune and inflammatory diseases. IL-21 is produced mainly from CD4+ T cells-in particular, follicular helper T (Tfh) cells-which have a great influence on the regulation of antibody production. It is also an important cytokine for the activation of CD8+ T cells, and its role in recovering the function of CD8+ T cells exhausted by chronic microbial infections and cancer has been clarified. Thus, IL-21 plays an extremely important role in viral infections, especially chronic viral infections. In this review, I will introduce the findings to date on how IL-21 is involved in some typical viral infections and the potential of treating viral diseases with IL-21.

Developmental retardation in neonates of aldehyde reductase (AKR1A)-deficient mice is associated with low ascorbic acid and high corticosterone levels.

Aldehyde reductase encoded by the Akr1a gene catalyzes the NADPH-dependent reduction of a variety of aldehyde compounds, and it plays a role in the biosynthesis of ascorbic acid (AsA) by converting D-glucuronate to L-gulonate. Although supplementing drinking water with AsA (1.5 mg/mL) ameliorates the fertility of Akr1a-/- (KO) female mice, litter sizes in the KO mice are typically smaller than those for Akr1a+/+ (WT) mice, and about one-third of the neonates have a reduced stature. Half of the neonates in the smallest, developmentally retarded group died before weaning, and the remaining half (less than 6 g in weight) also barely grew to adulthood. While no difference was found in the number of fetuses between the KO and WT mice at 14.5-embryonic days, the sizes of the KO fetuses had already diverged. Among the organs of these retarded KO neonates at 30 d, the spleen and thymus were characteristically small. While an examination of spleen cells showed the normal proportion of immune cells, apoptotic cell death was increased in the thymus, which would lead to thymic atrophy in the retarded KO neonates. Plasma AsA levels were lower in the small neonates despite the fact that their mothers had received sufficient AsA supplementation, and the corticosterone levels were inversely higher compared to wild-type mice. Thus, insufficient AsA contents together with a defect in corticosterone metabolism might be the cause of the retarded growth of the AKR1A-deficient mice embryos and neonates.

Physiological heating augments the anti-inflammatory reactions during granulocyte/monocyte apheresis: A in vitro study.

Granulocyte and monocyte adsorptive apheresis (GMA), an effective therapy for inflammatory disorders, exerts an anti-inflammatory influence by utilizing the biological reaction between blood and cellulose acetate (CA) beads, which are the carriers of the GMA column. Although the biological reaction has an optimum temperature, blood contacts the CA beads below body temperature as GMA is performed in an extracorporeal circulation system. We investigated various soluble factors in blood treated with CA beads at 25°C and 37°C. Here, the optimal temperature for IL-1 receptor antagonist (IL-1ra) release induced by CA beads was 37°C, and IL-6 production from monocytic cells was inhibited by the addition of plasma prepared from the CA bead-treated blood at 37°C, rather than at 25°C. These results indicated that physiological heating of the apheresis carrier augmented the anti-inflammatory reaction in vitro. Thus, heating during GMA may be a new approach for augmenting clinical efficacy.

Defective biosynthesis of ascorbic acid in Sod1-deficient mice results in lethal damage to lung tissue.

Superoxide dismutase 1 (Sod1) plays pivotal roles in antioxidation via accelerating the conversion of superoxide anion radicals into hydrogen peroxide, thus inhibiting the subsequent radical chain reactions. While Sod1 deficient cells inevitably undergo death in culture conditions, Sod1-knockout (KO) mice show relatively mild phenotypes and live approximately two years. We hypothesized that the presence of abundant levels of ascorbic acid (AsA), which is naturally produced in mice, contributes to the elimination of reactive oxygen species (ROS) in Sod1-KO mice. To verify this hypothesis, we employed mice with a genetic ablation of aldehyde reductase (Akr1a), an enzyme that is involved in the biosynthesis of AsA, and established double knockout (DKO) mice that lack both Sod1 and Akr1a. Supplementation of AsA (1.5 mg/ml in drinking water) was required for the DKO mice to breed, and, upon terminating the AsA supplementation, they died within approximately two weeks regardless of age or gender. We explored the etiology of the death from pathophysiological standpoints in principal organs of the mice. Marked changes were observed in the lungs in the form of macroscopic damage after the AsA withdrawal. Histological and immunological analyses of the lungs indicated oxidative damage of tissue and activated immune responses. Thus, preferential oxidative injury that occurred in pulmonary tissues appeared to be primary cause of the death in the mice. These collective results suggest that the pivotal function of AsA in coping with ROS in vivo, is largely in pulmonary tissues that are exposed to a hyperoxygenic microenvironment.

Analysis of intestinal immunity and flora in a collagen-induced mouse arthritis model: differences during arthritis progression.

Intestinal immunity and flora are reported to be associated with the onset of rheumatoid arthritis. However, differences in the intestinal immunity and flora dynamics between the initial peak and relapse of arthritis have not been investigated. Here we analyzed the lymphocyte populations in different lymphoid tissues, the IgA in feces, and the intestinal flora at the initial peak and the relapse phase of arthritis in a collagen-induced arthritis (CIA) mouse model. In this model compared with the control group, the percentage of RORγt+CD4+ T cells in the mesenteric lymph nodes (mLN) was increased at the initial peak but decreased at the relapse stage of arthritis, and the opposite changes were observed in the spleen. The percentage of Foxp3+CD4+ T cells was unchanged at the initial peak in both tissues but increased only in the mLN at the relapse stage. The IgA in feces increased with the progression of arthritis, and bacterial analysis revealed that some specific bacterial families were changed at the peak and relapse stages of arthritis. Finally, the immune dynamics under different arthritic conditions were examined by integrating these factors using principal component analysis (PCA). PCA showed that the immunological and intestinal flora profiles were different between the initial peak and the relapse of the arthritis. Our findings suggest that the intestinal immunity and the environment change drastically with the progress of arthritis.

GPI-80 as a Useful Index for Myeloid Cell Heterogeneity and a Potential Prognostic Biomarker for Metastatic Renal Cell Carcinoma.

Myeloid-derived suppressor cells (MDSCs), which include neutrophilic MDSCs and monocytic MDSCs, exhibit high immunosuppressive activity. Glycosylphosphatidylinositol-anchored 80 kD protein (GPI-80) is selectively expressed on mature neutrophils in healthy individuals. Increased GPI-80 expression on monocytes and variations in GPI-80 expression on neutrophils indicate the appearance of MDSCs in the peripheral blood of cancer patients. However, it is still unclear whether GPI-80 expression on myeloid cells, neutrophilic MDSCs and monocytic MDSCs, is correlated with the clinical outcomes of patients with cancer. In this study, we investigated the characteristics of myeloid cells expressing GPI-80 and the implication of GPI-80 expression in the clinical outcomes of patients with metastatic renal cell carcinoma (mRCC), in which primary renal cell carcinoma spreads from the kidney to other organs. The study included 20 patients with mRCC (a mean age of 66.0 years) and 16 healthy volunteers (a mean age of 47.8 years). To determine the heterogeneity of myeloid cells in peripheral blood samples, we performed the three-dimensional principal component analysis using the combination of GPI-80, CD16, and latency-associated peptide-1 (LAP), derived from the N-terminal region of transforming growth factor-ß1 precursor. The results showed that myeloid cells in mRCC patients were widely distributed and clearly distinguishable from those in the healthy volunteers. The survival analysis revealed that GPI-80 expression on neutrophils and monocytes was correlated with poor prognostic outcomes of patients with mRCC. In conclusion, the expression of GPI-80 on myeloid cells, a useful index for the heterogeneity of MDSCs, serves as a potential prognostic biomarker for mRCC.

Heterozygous SOD1 deficiency in mice with an NZW background causes male infertility and an aberrant immune phenotype.

New Zealand white (NZW) mouse is a mutant strain that has a larval defect in the immune system, and a F1 hybrid between NZW and New Zealand Black mouse spontaneously develops systemic lupus erythematosus (SLE). In meantime, the ablation of superoxide dismutase 1 (Sod1) causes autoimmune haemolytic anaemia, a clinical condition of SLE, in mice with a C57BL/6 background. On the basis of our previous studies, we hypothesised that oxidative stress may trigger this aberrant autoimmunity in NZW mice without crossing with another strain. To validate this, we attempted to establish Sod1-/-/NZW mice but this attempt failed to obtain any objective mouse. The congenic Sod1+/-/NZW male mice were completely infertile because of severe oligozoospermia attributed to a defect in spermatogenesis. The levels of the SOD1 protein were about a half in the testes of the Sod1+/-/NZW mice. Sperm from the Sod1+/-/NZW mice were largely defective and showed quite low fertilising ability in in vitro fertilisation assays. Concomitant with an increase in the oxidatively modified proteins, spermatogenic cells underwent more cell death in the testes of the Sod1+/-/NZW mice compared to those of WT/NZW mice. An examination of immunocompetent cells from Sod1+/-/NZW mice indicated an abnormality in T-cell responses. These collective results suggest that the oxidative stress caused by an SOD1 haploinsufficiency exerts deleterious effects on the testis, either directly on spermatogenic cells or via the destabilisation of the autoimmune response in Sod1+/-/NZW mice.

Interleukin-12p40 variant form reduces Interleukin-12p80 secretion.

IL-12 is a key cytokine for the promotion of CD4+ T cells differentiation to type 1 helper T cells. IL-12 is a heterodimer (IL-12p70) consisting of p40 and p35 subunits, and is mainly secreted from activated antigen-presenting cells, such as macrophages and dendritic cells (DCs). In this study, we found that activated mouse bone marrow-derived DCs (BMDCs) produced a p40 splice variant form mRNA in addition to the conventional p40 mRNA. This p40 variant mRNA was produced by alternative splicing in exon 5, and possessed a premature stop codon. As a result, the p40 variant protein contained 157 amino acids of the N-terminal part of p40 and an additional 10 novel amino acids. When the p40 variant was expressed in HEK-293T cells, it was not secreted from the cells. To investigate the function of the p40 variant, it was co-expressed with p40 and/or p35. The p40 variant did not affect the secretion of IL-12p40 or IL-12p70, or the function of the secreted p70. In contrast, the secretion of IL-12p80, a homodimeric IL-12 with two p40 subunits, was significantly decreased when the p40 variant was expressed. This new splicing variant p40 may act to fine-tune the function of IL-12p80.

IL-21 Enhances the Development of Colitis-Associated Colon Cancer: Possible Involvement of Activation-Induced Cytidine Deaminase Expression.

Inflammatory bowel diseases are known to be the origin of colitis-associated colon cancer (CAC). We previously reported that dextran sulfate sodium (DSS)-induced colitis is exacerbated in mouse-IL-21-isoform transgenic (Tg) mice. In this study, we assessed the CAC development induced by azoxymethane (AOM) and DSS in our Tg mice. AOM-DSS-induced tumor development was dramatically increased in the Tg mice compared with wild-type mice. IL-21 is known to enhance activation-induced cytidine deaminase (AID) expression in B cells and induce Ab class switching. In contrast, the AID expression in cells other than B cells initiates tumor development in many tissues. Therefore, we investigated whether IL-21 induces the AID expression in the large intestinal epithelial cells (IECs) during CAC development. AID gene and protein expression was increased in the IECs of AOM-DSS- or DSS-treated Tg mice compared with those of wild-type mice. Furthermore, we confirmed IL-21 induced AID gene expression in the purified IECs ex vivo. The present study also showed IL-21R gene expression in unstimulated wild-type mouse IECs, and this gene expression was augmented by TNF-α stimulation. The IL-21R expression and IL-21-induced AID gene activation were further confirmed in the Colon-38 cell line. Taken together, IL-21 may be involved in increasing the risk of CAC by enhancing the AID expression in IECs.

Runx1 and RORγt Cooperate to Upregulate IL-22 Expression in Th Cells through Its Distal Enhancer.

IL-22 is a cytokine that plays a pivotal role in regulating tissue homeostasis at barrier surfaces and is produced by activated CD4+ Th cells. Currently, the molecular mechanisms regulating Il22 gene expression are still unclear. In this study, we have identified a crucial cis-regulatory element located 32 kb upstream of the mouse Il22 promoter, termed conserved noncoding sequence (CNS)-32. We demonstrated that CNS-32 acts as an enhancer in reporter assays and contains binding motifs for Runt-related transcription factor (Runx)1 and retinoic acid-related orphan receptor γt (RORγt). Mutation of these motifs significantly abrogated the reporter activity, suggesting a role for both factors in the control of enhancer-mediated Il22 expression. Runx1 and RORγt occupancy and elevated histone H4 acetylation at CNS-32 were evident, as naive T cells differentiated into IL-22-producing Th22 cells. Overexpression of Runx1 promoted IL-22 production by inducing RORγt and IL-23 receptor, all critical to Th22 cell induction. Although Runx1 alone enhanced IL-22 production in Th22 cells, it was further enhanced in the presence of RORγt. Conversely, short hairpin RNA-mediated knockdown of core-binding factor ß, a cofactor essential for Runx1 activity, was effective in limiting IL-22 production. Collectively, our results suggest that IL-22 production is controlled by a regulatory circuit in which Runx1 induces RORγt and then partners with RORγt to direct Il22 expression through their targeting of the Il22 enhancer.

An Analysis of the Intracellular Signal Transduction of Peripheral Blood Leukocytes in Animal Models of Diabetes Using Flow Cytometry.

Various complications of diabetes are induced by the augmentation of chronic inflammation and attenuation of immunity. Leukocytes, which play major roles in inflammation and immune responses, are affected by the glycemic status and blood insulin level. In this chapter, we explain a method for analyzing the signal transduction pathway of leukocytes in peripheral blood. This method using flow cytometry can analyze a small amount of blood (50-100 µL/sample) without leukocyte purification. Thus, this procedure is useful for experiments using small-animal models of diabetes, such as mice and rats. We also introduce a new method for classifying intracellular signal transduction by combining the dispersibility level and the activation level of the signaling molecules.

Systemic neutrophil migration and rapid consumption of neutrophils in the spleen.

The systemic migration of neutrophils is not fully understood. In this study, we purified neutrophils from rat peripheral blood and labeled them with [51Cr] sodium chromate. The labeled cells were injected into the tail veins of rats, and were traced. Neutrophils were rapidly trapped in the liver and the spleen within 6 h. The migration ratios of neutrophils in the lung and the gut were lower compared with those in the liver and the spleen. Interestingly, migrated cells into the spleen were rapidly phagocytosed by monocytes/macrophages. Therefore, accumulation of intact neutrophils in the spleen may be difficult to measure.

Augmentation of the expression of the eotaxin receptor on duodenal neutrophils by IL-21.

Inflammation can occur via different mechanisms, such as via acute and chronic responses, on numerous occasions and function accordingly through various roles. There are more than five subsets of neutrophils; neutrophilic heterogeneity is modulated by the inflammatory condition. To understand the characteristics of inflammation, identification of atypical neutrophils is important. In this study, we found that the expression of eotaxin receptor (CD193) on atypical neutrophils in the duodenum is augmented in IL-21 isoform transgenic (Tg) mice. In a series of studies, we have established a Tg mouse strain to further investigate the functions of IL-21 in vivo. Interestingly, Tg mice immunized with ovalbumin (OVA) were more sensitive to OVA-induced systemic anaphylaxis as compared with wild type mice with duodenal and splenic gross congestion. Further analysis conducted in the duodenum of Tg mice revealed that only the number of neutrophils migrating into the duodenum was significantly increased prior to immunization. Previous studies have shown that the gastrointestinal compartment and the spleen constantly produce eotaxin, which regulates basal levels of tissue eosinophils. Therefore, we analyzed CD193 expression on neutrophils and eosinophils. As expected, its expression by duodenal neutrophils was upregulated in Tg mice. Furthermore, the addition of IL-21 into bone marrow cell culture increased the number of CD193+ neutrophils, which easily migrated into the duodenum. These observations suggested that CD193+ neutrophils increase in number under inflammatory conditions due to chronic IL-21 production.

Adsorption of Soluble Immunoglobulin-Type Adhesion Molecules to Cellulose Acetate Beads.

Circulating levels of soluble intercellular adhesion molecule-1 (sICAM-1) and vascular adhesion molecule-1 (sVCAM-1) are elevated in patients with inflammatory bowel disease. Cellulose acetate (CA) beads are used as carriers for granulocyte and monocyte (GM) adsorptive apheresis (GMA). We investigated the effect of CA beads on sICAM-1 and sVCAM-1 plasma concentrations in vitro. Because GM adsorption to CA beads increased with a rise in the incubation temperature in our previous study, peripheral blood was incubated with and without CA beads at 5, 25, 37, or 43 °C and plasma sICAM-1 and sVCAM-1 was measured. The sICAM-1 and sVCAM-1 concentrations in samples incubated with CA beads were significantly lower than those without CA beads at all four temperatures. However, no significant differences were observed both sICAM-1 and sVCAM-1 plasma levels at the four different temperatures after incubation with CA beads. These results suggest that independent of incubation temperature, sICAM-1 and sVCAM-1 are likely to adsorb CA beads. These molecules may be a new index for predicting the therapeutic effects of GMA.

Protein Tyrosine Phosphatase SHP-2 Is Involved in the Interleukin-21-Induced Activation of Extracellular Signal-Regulated Kinase 1/2.

The cytokine interleukin-21 (IL-21) is mainly produced from activated CD4+ T cells and natural killer T (NKT) cells. IL-21 enhances the proliferation and differentiation of T cells and B cells and also increases cytotoxicity of CD8+ T cells and NK cells through the IL-21 receptor and its downstream signaling molecules such as signal transducers and activator of transcription 3 (STAT3) and extracellular signal-regulated kinase 1/2 (ERK1/2). SH2 domain-containing tyrosine phosphatase (SHP-2) is ubiquitously expressed, including hematopoietic cells. SHP-2 has been implicated in the regulation of IL-6 and IL-3 signaling, but its function in IL-21 signaling has not been investigated. Therefore, we studied the role of SHP-2 in IL-21 signaling by SHP-2 overexpression and knockdown experiments. For the SHP-2 overexpression, we used 293T human embryonic kidney cells, in which the IL-21 receptor system were easily reconstituted and high amounts of exogenous SHP-2 were expressed by vector transfection. In 293T cells, overexpressed SHP-2 caused the increase in the degree of the IL-21-induced ERK1/2 activation. Subsequently, SHP-2 knockdown experiments were performed in the mouse pro-B cell line, BAF21RWT-1, which constitutively expresses human IL-21 receptor and proliferates in an IL-21-dependent manner. SHP-2 knockdown reduced the degree of the IL-21-induced ERK1/2 activation and suppressed cell proliferation. These results suggest that SHP-2 may augment the ERK1/2 activity and cell proliferation activity in IL-21 signaling. We propose that SHP-2 is involved in the IL-21-mediated ERK1/2 activation and cell proliferation.

IL-21 Attenuates FITC-Induced Contact Hypersensitivity Response via Regulation of Dendritic Cell Function.

IL-21 is mainly produced by activated CD4+ T cells and is involved in the activation of immune cells such as T cells and macrophages. In contrast, IL-21 suppresses dendritic cell maturation. We studied the effect of IL-21 in a mouse model of FITC-induced contact hypersensitivity using IL-21 isoform transgenic (IL-21iso-Tg) mice. Tissue inflammation at 24 hours after elicitation in IL-21iso-Tg mice was significantly weaker than that in wild-type mice. In agreement with tissue inflammation, recruitment of CD4+ and CD8+ T cells, neutrophils, and macrophages into the inflamed tissue was decreased in IL-21iso-Tg mice. In addition, both mRNA expression and protein production of inflammatory cytokines were lower in IL-21iso-Tg mice. In the skin, T cells were activated at inducible skin-associated lymphoid tissue, which is likely a gut-associated lymphoid tissue. The mRNA level of CXCL2, an essential chemokine for inducible skin-associated lymphoid tissue formation, was significantly lower in IL-21iso-Tg mice, and histological analysis showed that dendritic cell clustering, a preliminary step in inducible skin-associated lymphoid tissue formation, was impaired. Our study showed that IL-21 down-regulated inducible skin-associated lymphoid tissue formation and reduced contact hypersensitivity response.