MUTATIONS IN STAT3 ASSOCIATED WITH HUMAN HYPER IgE SYNDROME ENHANCE NFκB AND MAPK-MEDIATED GENE EXPRESSION

Hyper IgE syndrome (HIES) is a multisystem disorder resulting in bone and immune system abnormalities. It is associated with mutations in STAT3, which disrupt protein domains responsible for transcriptional function. Patients with HIES display osteoporosis and enhanced inflammatory cytokine production similar to hematopoietic Stat3-deficient mice. Since osteoclast and inflammatory cytokine genes are NFκB targets, these observations indicate a possible deregulation of NFκB signaling in both mice and humans with STAT3-deficiency. Here, we sought to examine the role of STAT3 in the regulation of NFκB-mediated gene expression through analysis of three HIES STAT3 point mutations in both hematopoietic and non- hematopoietic cells. We found that IL-6-induced tyrosine phosphorylation of STAT3 was partially or completely abrogated by HIES mutations in the transactivation domain (V713L) or SH2 domain (V637M), respectively, in both hematopoietic and non- hematopoietic cells. By contrast, IL-6-induced tyrosine phosphorylation of an HIES mutant in the STAT3 DNA-binding domain (R382W) was intact. The R382W and V713L mutants significantly reduced IL-6-dependent STAT3 transcriptional activity in reporter gene assays. Moreover, the R382W and V637M mutants significantly diminished IL-6-responsive expression of the endogenous STAT3 target gene, Socs3, as assessed by quantitative real-time PCR (qPCR) in the RAW macrophage cell line. These observations indicate the HIES mutants dominantly suppress the transcriptional activity of wild type STAT3, albeit to varying degrees. All three HIES mutants enhanced LPS-induced expression of the NFκB target genes IL6 (IL-6), Cxcl10 (IP- 10), and Tnf (TNFα) in RAW cells, as indicated by qPCR. Furthermore, overexpression of wild type STAT3 in Stat3-deficient murine embryonic fibroblasts significantlyreduced LPS-stimulated expression of IL6, Cxcl10, and IL12p35. In addition, in aprimary murine osteoclast differentiation assay, a STAT3-specific SH2 domain inhibitor led to significantly increased levels of osteoclast-specific gene expression. These results suggest that STAT3 serves as a negative regulator of NFκB-mediated gene expression, and furthermore imply that STAT3 mutations associated with HIES contribute to the osteopenia and inflammation observed in HIES patients.

Macrophage inflammatory protein-2: Chromosomal regulation in rat small intestinal epithelial cells

Nonpathogenic, resident bacteria participate in the pathogenesis of inflammation in the small intestine, but the molecular messages produced by such bacteria are unknown. Inflammatory responses involve the recruitment of specific leukocyte subsets. We, therefore, hypothesized that butyrate, a normal bacterial metabolite, may modulate chemokine secretion by epithelial cells, by amplifying their response to proinflammatory signals. We studied the expression of the chemokine, macrophage inflammatory protein-2 (MIP-2) by the rat small intestinal epithelial cell line, IEC-6. Cells were stimulated with lipopolysaccharide or with interleukin 1β (IL-1β) and incubated with sodium butyrate. Acetylation of histones was examined in Triton X acetic acid–urea gels by PAGE. Unstimulated IEC-6 cells did not secrete MIP-2. However, lipopolysaccharide and IL-1β induced MIP-2 expression. Butyrate enhanced MIP-2 secretion both in lipopolysaccharide-stimulated and IL-1β-stimulated enterocytes; but butyrate alone did not induce MIP-2 expression. Butyrate increased the acetylation of histones extracted from the nuclei of IEC-6 cells. Furthermore, acetylation of histones (induced by trichostatin A, a specific inhibitor of histone deacetylase) enhanced MIP-2 expression by cells stimulated with IL-1β. In conclusion, trichostatin A reproduced the effects of butyrate on MIP-2 secretion. Butyrate, therefore, increases MIP-2 secretion in stimulated cells by increasing histone acetylation. We speculate that butyrate carries information from bacteria to epithelial cells. Epithelial cells transduce this signal through histone deacetylase, modulating the secretion of chemokines.

Stress-induced phosphorylation of STAT1 at Ser727 requires p38 mitogen-activated protein kinase whereas IFN-γ uses a different signaling pathway

STAT1 is an essential transcription factor for macrophage activation by IFN-γ and requires phosphorylation of the C-terminal Ser727 for transcriptional activity. In macrophages, Ser727 phosphorylation in response to bacterial lipopolysaccharide (LPS), UV irradiation, or TNF-α occurred through a signaling path sensitive to the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 whereas IFN-γ-mediated Ser727 phosphorylation was not inhibited by the drug. Consistently, SB203580 did not affect IFN-γ-mediated, Stat1-dependent transcription but inhibited its enhancement by LPS. Furthermore, LPS, UV irradiation, and TNF-α caused activation of p38 MAPK whereas IFN-γ did not. An essential role for p38 MAPK activity in STAT1 Ser727 phosphorylation was confirmed by using cells expressing an SB203580-resistant p38 MAPK. In such cells, STAT1 Ser727 phosphorylation in response to UV irradiation was found to be SB203580 insensitive. Targeted disruption of the mapkap-k2 gene, encoding a kinase downstream of p38 MAPK with a key role in LPS-stimulated TNF-α production and stress-induced heat shock protein 25 phosphorylation, was without a significant effect on UV-mediated Ser727 phosphorylation. The recombinant Stat1 C terminus was phosphorylated in vitro by p38MAPKα and β but not by MAPK-activated protein kinase 2. Janus kinase 2 activity, previously reported to be required for IFN-γ-mediated Ser727 phosphorylation, was not needed for LPS-mediated Ser727 phosphorylation, and activation of Janus kinase 2 did not cause the appearance of STAT1 Ser727 kinase activity. Our data suggest that STAT1 is phosphorylated at Ser727 by a stress-activated signaling pathway either through p38 MAPK directly or through an unidentified kinase downstream of p38MAPK.

Exit from G0 and entry into the cell cycle of cells expressing p21Sdi1 antisense RNA.

p21Sdi1 (also known as Cip1 and Waf1), an inhibitor of DNA synthesis cloned from senescent human fibroblasts, is an inhibitor of G1 cyclin-dependent kinases (Cdks) in vitro and is transcriptionally regulated by wild-type p53. In addition, p21Sdi1 has been found to inhibit DNA replication by direct interaction with proliferating cell nuclear antigen. In this study we analyzed normal human fibroblast cells arrested in G0 and determined that an excess of p21Sdi1 was present after immunodepletion of various cyclins and Cdks, in contrast to mitogen-stimulated cells in early S phase. Expression of antisense p21Sdi1 RNA in G0-arrested cells resulted in induction of DNA synthesis as well as entry into mitosis. These results suggest that p21Sdi1 functions in G0 and early G1 and that decreased expression of the gene is necessary for cell cycle progression.

Transglutaminase 2 null macrophages respond to lipopolysaccharide stimulation by elevated proinflammatory cytokine production due to an enhanced αvβ3 integrin-induced Src tyrosine kinase signaling

Transglutaminase 2 (TG2) is a protein crosslinking enzyme with several additional biochemical functions. Loss of TG2 in vivo results in impaired phagocytosis of apoptotic cells and altered proinflammatory cytokine production by macrophages engulfing apoptotic cells leading to autoimmunity. It has been proposed that TG2 acts as an integrin ß(3) coreceptor in the engulfment process, while altered proinflammatory cytokine production is related to the lack of latent TGFß activation by TG2 null macrophages. Here we report that TG2 null macrophages respond to lipopolysaccharide treatment by elevated IL-6 and TNFa production. Though TGFß has been proposed to act as a feed back regulator of proinflammatory cytokine production in LPS-stimulated macrophages, this phenomenon is not related to the lack of active TGFß production. Instead, in the absence of TG2 integrin ß(3) maintains an elevated basal Src family kinase activity in macrophages, which leads to enhanced phosphorylation and degradation of the I?Ba. Low basal levels of I?Ba explain the enhanced sensitivity of TG2 null macrophages to signals that regulate NF-?B. Our data suggest that TG2 null macrophages bear a proinflammatory phenotype, which might contribute to the enhanced susceptibility of these mice to develop autoimmunity and atherosclerosis.

Wild mushrooms and their mycelia as sources of bioactive compounds: antioxidant, anti-inflammatory and cytotoxic properties

Mushrooms are an important source of natural compounds with acknowledged bioactivity. Pleurotus eryngii (DC.) Quél., in particular, is widely recognized for its organoleptic quality and favorable health effects, being commercially produced in great extent. On the other hand, Suillus bellinii (Inzenga) Watling is an ectomycorrhizal symbiont, whose main properties were only reported in a scarce number of publications. Some current trends point toward using the mycelia and the culture media as potential sources of bioactive compounds, in addition to the fruiting bodies. Accordingly, P. eryngii and S. bellinii were studied for their composition in phenolic acids and sterols, antioxidant capacity (scavenging DPPH radicals, reducing power, β-carotene bleaching inhibition and TBARS formation inhibition), anti-inflammatory effect (by down-regulating LPS-stimulated NO in RAW264.7 cells) and anti-proliferative activity (using MCF-7, NCI-H460, HeLa, HepG2 and PLP2 cell lines). Overall, S. bellinii mycelia showed higher contents of ergosterol and phenolic compounds (which were also detected in higher quantity in its fruiting body) and stronger antioxidant activity than P. eryngii. On the other hand, P. eryngii mycelia showed anti-inflammatory (absent in S. bellinii mycelia) and a cytotoxicity similar (sometimes superior) to its fruiting bodies, in opposition to S. bellinii, whose mycelia presented a decreased anti-proliferative activity. Furthermore, the assayed species showed differences in the growth rate and yielded biomass of their mycelia, which should also be considered in further applications.

Anti-oxidative and anti-neuroinflammatory effects of ethyl acetate extract fractions of Suaeda asparagoides MIq

Purpose: To evaluate the in vitro antioxidant and anti-neuroinflammatory effects of Suaeda asparagoides ethylacetate extract (SAE) in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. Methods: The antioxidative activity of SAE was evaluated by measuring 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging activity spectrometrometrically. Cell viability was evaluated by 3-(4, 5dimethylthiazol-2-yl)-2, 5- diphenyl-tetrazolium bromide (MTT) assay, while LPS-stimulated BV-2 microglia were used to study the expression and production of inflammatory mediators, including nitric oxide (NO), inducible NO synthase (iNOS) and tumor necrosis alpha (TNF-α). Results: Pretreatment with SAE prior to LPS treatment significantly inhibited excessive production of NO (p < 0.001 at 20, 40, 80 and 100 μg/mL) in a dose-dependent manner, and was associated with down-regulation of expression of inducible nitric oxide synthase (iNOS). SAE also suppressed the LPSinduced increase in TNF-α level (p < 0.01at concentrations of 40 and 80 μg/mL) in BV-2 cells. Furthermore, DPPH-generated free radicals were inhibited by SAE in a concentration-dependent manner. Conclusion: These results indicate that SAE possesses strong anti-oxidant properties, and inhibits excessive production of pro-inflammatory mediators, including NO, iNOS and TNF-α, in LPS-stimulated BV-2 cells

Modulation of peritoneal macrophage activity by the saturation state of the fatty acid moiety of phosphatidylcholine

To determine the effects of saturated and unsaturated fatty acids in phosphatidylcholine (PC) on macrophage activity, peritoneal lavage cells were cultured in the presence of phosphatidylcholine rich in saturated or unsaturated fatty acids (sat PC and unsat PC, respectively), both used at concentrations of 32 and 64 µM. The treatment of peritoneal macrophages with 64 µM unsat PC increased the production of hydrogen peroxide by 48.3% compared to control (148.3 ± 16.3 vs 100.0 ± 1.8%, N = 15), and both doses of unsat PC increased adhesion capacity by nearly 50%. Moreover, 64 µM unsat PC decreased neutral red uptake by lysosomes by 32.5% compared to the untreated group (67.5 ± 6.8 vs 100.0 ± 5.5%, N = 15), while both 32 and 64 µM unsat PC decreased the production of lipopolysaccharide-elicited nitric oxide by 30.4% (13.5 ± 2.6 vs 19.4 ± 2.5 µM) and 46.4% (10.4 ± 3.1 vs 19.4 ± 2.5 µM), respectively. Unsat PC did not affect anion production in non-stimulated cells or phagocytosis of unopsonized zymosan particles. A different result pattern was obtained for macrophages treated with sat PC. Phorbol 12-miristate 13-acetate-elicited superoxide production and neutral red uptake were decreased by nearly 25% by 32 and 64 µM sat PC, respectively. Sat PC did not affect nitric oxide or hydrogen peroxide production, adhesion capacity or zymosan phagocytosis. Thus, PC modifies macrophage activity, but this effect depends on cell activation state, fatty acid saturation and esterification to PC molecule and PC concentration. Taken together, these results indicate that the fatty acid moiety of PC modulates macrophage activity and, consequently, is likely to affect immune system regulation in vivo.

Modulation of human neutrophil oxidative metabolism and degranulation by extract of Tamarindus indica L. fruit pulp

The tamarind (Tamarindus indica L) is indigenous to Asian countries and widely cultivated in the American continents. The tamarind fruit pulp extract (ExT), traditionally used in spices, food components and juices, is rich in polyphenols that have demonstrated anti-atherosclerotic, antioxidant and immunomodulatory activities. This study evaluated the modulator effect of a crude hydroalcoholic ExT on some peripheral human neutrophil functions. The neutrophil reactive oxygen species generation, triggered by opsonized zymosan (OZ), n-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA), and assessed by luminol- and lucigenin-enhanced chemiluminescence (LumCL and LucCL, respectively), was inhibited by ExT in a concentration-dependent manner. ExT was a more effective inhibitor of the PMA-stimulated neutrophil function [IC(50) (in mu g/10(6)cells) = 115.7 +/- 9.7 (LumCL) and 174.5 +/- 25.9 (LucCL)], than the OZ- [IC(50) = 248.5 +/- 23.1 (LumCL) and 324.1 +/- 34.6 (LucCL)] or fMLP-stimulated cells [IC(50) = 178.5 +/- 12.2 (LumCL)]. The ExT also inhibited neutrophil NADPH oxidase activity (evaluated by O(2) consumption), degranulation and elastase activity (evaluated by spectrophotometric methods) at concentrations higher than 200 mu g/10(6) cells, without being toxic to the cells, under the conditions assessed. Together, these results indicate the potential of ExT as a source of compounds that can modulate the neutrophil-mediated inflammatory diseases. (C) 2008 Elsevier Ltd. All rights reserved.

Chemokine and cell adhesion molecule mRNA expression and neutrophil infiltration in lipopolysaccharide-induced hepatitis in ethanol-fed rats

Neutrophil infiltration is a feature of alcoholic hepatitis (AH), and although the mechanism by which this occurs is unclear, it may involve a chemotactic gradient. We used lipopolysaccharide (LPS) to induce, in ethanol-fed rats, liver damage similar to that seen in AH. To our knowledge, this study is the first to examine the effect of ethanol on LPS-stimulated chemokine mRNA expression in this model. Hepatic cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-2, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1 beta, MIP-2, and eotaxin mRNA levels were elevated 1 to 3 hr post-LPS in both groups. Maximal expression of MIP-2 and MCP-1 mRNA was higher in ethanol-fed rats 1 hr post-LPS, whereas CINC-2 mRNA expression was elevated above controls at 12 to 24 hr. Hepatic intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 mRNA levels were elevated in both groups at 1 hr, whereas L-selectin expression in ethanol-fed rats was elevated above controls at 12 to 24 hr. Hepatic neutrophil infiltration was highest during maximal hepatocyte necrosis. These data suggest that cell adhesion molecules, in conjunction with elevated cytokines and the subsequently induced chemokines, may assist in the formation of a chemotactic gradient within the liver, causing the neutrophil infiltration seen both in this model and possibly in AH.

PPAR gamma expression is increased in systemic lupus erythematosus patients and represses CD40/CD40L signaling pathway

Systemic lupus erythematosus (SLE) is a heterogeneous disease involving several immune cell types and pro-inflammatory signals, including the one triggered by binding of CD40L to the receptor CD40. Peroxisome-proliferator activated receptor gamma (PPAR gamma) is a transcription factor with anti-inflammatory properties. Here we investigated whether CD40 and PPAR gamma could exert opposite effects in the immune response and the possible implications for SLE. Increased PPAR gamma mRNA levels were detected by real-time PCR in patients with active SLE, compared to patients with inactive SLE PPAR gamma/GAPDH mRNA = 2.21 +/- 0.49 vs. 0.57 +/- 0.14, respectively (p < 0.05) or patients with infectious diseases and healthy subjects (p < 0.05). This finding was independent of the corticosteroid therapy. We further explored these observations in human THP1 and in SLE patient-derived macrophages, where activation of CD40 by CD40L promoted augmented PPAR gamma gene transcription compared to non-stimulated cells (PPAR gamma/GAPDH mRNA = 1.14 +/- 0.38 vs. 0.14 +/- 0.01, respectively; p < 0.05). This phenomenon occurred specifically upon CD40 activation, since lipopolysaccharide treatment did not induce a similar response. In addition, increased activity of PPAR gamma was also detected after CD40 activation, since higher PPAR gamma-dependent transcription of CD36 transcription was observed. Furthermore, CD40L-stimulated transcription of CD80 gene was elevated in cells treated with PPAR gamma-specific small interfering RNA (small interfering RNA, siRNA) compared to cells treated with CD40L alone (CD80/GAPDH mRNA = 0.11 +/- 0.04 vs. 0.05 +/- 0.02, respectively; p < 0.05), suggesting a regulatory role for PPAR gamma on the CD40/CD40L pathway. Altogether, our findings outline a novel mechanism through which PPAR gamma regulates the inflammatory signal initiated by activation of CD40, with important implications for the understanding of immunological mechanisms underlying SLE and the development of new treatment strategies. Lupus (2011) 20, 575-587.

Effect of Isolated Fractions of Harpagophytum procumbens DC (Devil`s Claw) on COX-1, COX-2 Activity and Nitric Oxide Production on Whole-Blood Assay

The present study evaluates the effect of isolated fractions of Harpagophytum procumbens (devil`s claw) on cyclooxygenase (COX-1 and COX-2) activities and NO production using a whole blood assay. The activity of COX-1 was quantified as platelet thromboxane B(2) production in blood clotting and COX-2 as prostaglandin E(2) production in LPS-stimulated whole blood. Total NO(2)(-)/NO(3)(-) concentration was determined by Griess reaction in LPS stimulated blood. Assays were performed by incubation of isolated fractions obtained by flash chromatography monitored with HPLC, TLC and identified by (1)HNMR, containing different amounts of harpagoside with blood from healthy donors. Indomethacin and etoricoxib were the positive controls of COX-1 and COX-2 Inhibition. Data shows that fraction containing the highest concentration of harpagoside inhibited indistinctively COX-1 and COX-2 (37.2 and 29.5% respectively) activity and greatly inhibited NO production (66%). In contrast the fraction including iridoid pool increased COX-2 and did not alter NO and COX-1 activities. The fraction containing cinnamic acid was able to reduce only NO production (67%). Our results demonstrated that the harpagoside fraction is the main responsible for the effect of devils claw on these enzyme activities. However, other components from devil`s claw crude extract could antagonize or increase the synthesis of inflammatory mediators. Copyright (C) 2010 John Wiley & Sons, Ltd.

Tyrosines in the Carboxyl Terminus Regulate Syk Kinase Activity and Function

The Syk tyrosine kinase family plays an essential role in immunoreceptor tyrosine-based activation motif (ITAM) signaling. The binding of Syk to tyrosine-phosphorylated ITAM subunits of immunoreceptors, such as Fc epsilon RI on mast cells, results in a conformational change, with an increase of enzymatic activity of Syk. This conformational change exposes the COOH-terminal tail of Syk, which has three conserved Tyr residues (Tyr-623, Tyr-624, and Tyr-625 of rat Syk). To understand the role of these residues in signaling, wild-type and mutant Syk with these three Tyr mutated to Phe was expressed in Syk-deficient mast cells. There was decreased Fc epsilon RI-induced degranulation, nuclear factor for T cell activation and NF kappa B activation with the mutated Syk together with reduced phosphorylation of MAP kinases p38 and p42/44 ERK. In non-stimulated cells, the mutated Syk was more tyrosine phosphorylated predominantly as a result of autophosphorylation. In vitro, there was reduced binding of mutated Syk to phosphorylated ITAM due to this increased phosphorylation. This mutated Syk from non-stimulated cells had significantly reduced kinase activity toward an exogenous substrate, whereas its autophosphorylation capacity was not affected. However, the kinase activity and the autophosphorylation capacity of this mutated Syk were dramatically decreased when the protein was dephosphorylated before the in vitro kinase reaction. Furthermore, mutation of these tyrosines in the COOH-terminal region of Syk transforms it to an enzyme, similar to its homolog ZAP-70, which depends on other tyrosine kinases for optimal activation. In testing Syk mutated singly at each one of the tyrosines, Tyr-624 but especially Tyr-625 had the major role in these reactions. Therefore, these results indicate that these tyrosines in the tail region play a critical role in regulating the kinase activity and function of Syk.

Gas6 and its receptors are implicated in sepsis as modulators of innate immunity

Gas6 downregulates the activation state of macrophages and thereby their production of proinflammatory cytokines induced by various stimuli. We aimed to determine whether Gas6 is involved in sepsis. We measured Gas6 plasma levels in 13 healthy subjects, 29 patients with severe sepsis, and 18 patients with non-infectious inflammatory diseases. Gas6 level was higher in septic patients than in control groups (P 0.0001). The sensitivity and specificity of Gas6 levels to predict fatal outcome were 83% and 88%. We next investigated whether Gas6 affects cytokine production and outcome in experimental models of endotoxemia and peritonitis in wild-type (WT) and Gas6-/- mice. Circulating levels of Gas6 after LPS 25mg/kg i.p. peaked at 1 hour (P<0.001). Similarly, TNF- was higher in Gas6-/- than in WT mice 1 hour after LPS (P<0.05). Furthermore, 62 anti- and pro-inflammatory cytokines were quantified in plasma after LPS injection. Their levels were globally higher in Gas6-/- plasma after LPS, 47/62 cytokines being at least 50% higher in Gas6-/- than in WT plasma after 1 hour. Mortality induced by 25mg/kg LPS was 25% in WT versus 87% in Gas6-/- mice (P<0.05). LPS-induced mortality in Gas6 receptors Axl-/-, Tyro3-/- and Merkd was also enhanced when compared to WT mice (P<0.001). In peritonitis models (cecal ligation and puncture, CLP, and i.p. injection of E. coli), Gas6 plasma levels increased and remained elevated at least 24 hours. CLP increased mortality in Gas6-/- mice. Finally, we explored the role of Gas6 in LPS-treated macrophages. We found that Gas6 was released by LPS-stimulated WT macrophages and that Gas6-/- macrophages produced more TNF- and IL-6 than WT macrophages. Cytokine release by Gas6-/- macrophages was higher than by WT macrophages (cytokine array). Adjunction of recombinant Gas6 to the culture medium of Gas6-/- macrophages diminished the cytokine production to WT levels. In LPS-treated Gas6-/- macrophages, Akt and Erk1/2 phosphorylation was reduced whereas p38 and NF B activation was enhanced. Thus, in septic patients, elevated Gas6 levels were associated with fatal outcome. In mice, they raised in experimental endotoxemia and peritonitis models, and correlated also with sepsis severity. However, Gas6-/- mice survival in these models was reduced compared to WT. Gas6 secreted by macrophages in response to LPS activated Akt and restrained p38 and NF B activation, thereby dampening macrophage activation. Altogether these data suggest that, during endotoxemia, Gas6-/- mice phenotype resembles that of mice which have undergone PI3K inhibition, indicating that Gas6 is a major modulator of innate immunity.

Periodic fever, aphthous stomatitis, pharyngitis, cervical adenitis syndrome is linked to dysregulated monocyte IL-1β production.

BACKGROUND: The exact pathogenesis of the pediatric disorder periodic fever, aphthous stomatitis, pharyngitis, cervical adenitis (PFAPA) syndrome is unknown. OBJECTIVES: We hypothesized that PFAPA might be due to dysregulated monocyte IL-1β production linked to genetic variants in proinflammatory genes. METHODS: Fifteen patients with PFAPA syndrome were studied during and outside a febrile episode. Hematologic profile, inflammatory markers, and cytokine levels were measured in the blood. The capacity of LPS-stimulated PBMCs and monocytes to secrete IL-1β was assessed by using ELISA, and active IL-1β secretion was visualized by means of Western blotting. Real-time quantitative PCR was performed to assess cytokine gene expression. DNA was screened for variants of the MEFV, TNFRSF1A, MVK, and NLRP3 genes in a total of 57 patients with PFAPA syndrome. RESULTS: During a febrile attack, patients with PFAPA syndrome revealed significantly increased neutrophil counts, erythrocyte sedimentation rates, and C-reactive protein, serum amyloid A, myeloid-related protein 8/14, and S100A12 levels compared with those seen outside attacks. Stimulated PBMCs secreted significantly more IL-1β during an attack (during a febrile episode, 575 ± 88 pg/mL; outside a febrile episode, 235 ± 56 pg/mL; P < .001), and this was in the mature active p17 form. IL-1β secretion was inhibited by ZYVAD, a caspase inhibitor. Similar results were found for stimulated monocytes (during a febrile episode, 743 ± 183 pg/mL; outside a febrile episode, 227 ± 92 pg/mL; P < .05). Genotyping identified variants in 15 of 57 patients, with 12 NLRP3 variants, 1 TNFRSF1A variant, 4 MEFV variants, and 1 MVK variant. CONCLUSION: Our data strongly suggest that IL-1β monocyte production is dysregulated in patients with PFAPA syndrome. Approximately 20% of them were found to have NLRP3 variants, suggesting that inflammasome-related genes might be involved in this autoinflammatory syndrome.