Maternal satisfaction with administering infant interventions in the neonatal intensive care unit.

OBJECTIVE: To examine mothers' satisfaction with administering interventions for their preterm infants and with the helpfulness of the study nurse by comparing massage with auditory, tactile, visual, and vestibular stimulation (ATVV intervention), kangaroo care, and education about equipment needed at home and to explore whether mother and infant characteristics affected maternal satisfaction ratings. DESIGN: Three-group experimental design. SETTING: Four neonatal intensive care units (NICUs) (two in North Carolina, two in Illinois). PARTICIPANTS: Two hundred and eight (208) preterm infants and their mothers. METHODS: When the infant was no longer critically ill, mother/infant dyads were randomly assigned to ATVV, kangaroo care, or the education group all taught by study nurses. At discharge and 2 months corrected age, mothers completed questionnaires. RESULTS: All groups were satisfied with the intervention and with nurse helpfulness, and the degree of satisfaction did not differ among them. Intervention satisfaction, but not nurse helpfulness, was related to recruitment site. Older, married, and minority mothers were less satisfied with the intervention but only at 2 months. Higher anxiety was related to lower intervention satisfaction at discharge and lower ratings of nurse helpfulness at discharge and 2 months. More depressive symptoms were related to lower nurse helpfulness ratings at 2 months. CONCLUSIONS: Mothers were satisfied with interventions for their infants regardless of the intervention performed. Maternal satisfaction with the intervention was related to recruitment site, maternal demographic characteristics, and maternal psychological distress, especially at 2 months. Thus, nursing interventions that provide mothers with a role to play in the infant's care during hospitalization are particularly likely to be appreciated by mothers.

Acute reactogenicity after intramuscular immunization with recombinant vesicular stomatitis virus is linked to production of IL-1ß.

Vaccines based on live viruses are attractive because they are immunogenic, cost-effective, and can be delivered by multiple routes. However, live virus vaccines also cause reactogenic side effects such as fever, myalgia, and injection site pain that have reduced their acceptance in the clinic. Several recent studies have linked vaccine-induced reactogenic side effects to production of the pro-inflammatory cytokine interleukin-1ß (IL-1ß) in humans. Our objective was therefore to determine whether IL-1ß contributed to pathology after immunization with recombinant vesicular stomatitis virus (rVSV) vaccine vectors, and if so, to identify strategies by which IL-1ß mediated pathology might be reduced without compromising immunogenicity. We found that an rVSV vaccine induced local and systemic production of IL-1ß in vivo, and that accumulation of IL-1ß correlated with acute pathology after rVSV immunization. rVSV-induced pathology was reduced in mice deficient in the IL-1 receptor Type I, but the IL-1R-/- mice were fully protected from lethal rechallenge with a high dose of VSV. This result demonstrated that IL-1 contributed to reactogenicity of the rVSV, but was dispensable for induction of protective immunity. The amount of IL-1ß detected in mice deficient in either caspase-1 or the inflammasome adaptor molecule ASC after rVSV immunization was not significantly different than that produced by wild type animals, and caspase-1-/- and ASC-/- mice were only partially protected from rVSV-induced pathology. Those data support the idea that some of the IL-1ß expressed in vivo in response to VSV may be activated by a caspase-1 and ASC-independent mechanism. Together these results suggest that rVSV vectors engineered to suppress the induction of IL-1ß, or signaling through the IL-1R would be less reactogenic in vivo, but would retain their immunogenicity and protective capacity. Such rVSV would be highly desirable as either vaccine vectors or oncolytic therapies, and would likely be better tolerated in human vaccinees.

Hyaluronan activation of the Nlrp3 inflammasome contributes to the development of airway hyperresponsiveness.

BACKGROUND: The role of the Nlrp3 inflammasome in nonallergic airway hyperresponsiveness (AHR) has not previously been reported. Recent evidence supports both interleukin (IL) 1ß and short fragments of hyaluronan (HA) as contributors to the biological response to inhaled ozone. OBJECTIVE: Because extracellular secretion of IL-1ß requires activation of the inflammasome, we investigated the role of the inflammasome proteins ASC, caspase1, and Nlrp3 in the biological response to ozone and HA. METHODS: C57BL/6J wild-type mice and mice deficient in ASC, caspase1, or Nlrp3 were exposed to ozone (1 ppm for 3 hr) or HA followed by analysis of airway resistance, cellular inflammation, and total protein and cytokines in bronchoalveolar lavage fluid (BALF). Transcription levels of IL-1ß and IL-18 were determined in two populations of lung macrophages. In addition, we examined levels of cleaved caspase1 and cleaved IL-1ß as markers of inflammasome activation in isolated alveolar macrophages harvested from BALF from HA-treated mice. RESULTS: We observed that genes of the Nlrp3 inflammasome were required for development of AHR following exposure to either ozone or HA fragments. These genes are partially required for the cellular inflammatory response to ozone. The expression of IL-1ß mRNA in alveolar macrophages was up-regulated after either ozone or HA challenge and was not dependent on the Nlrp3 inflammasome. However, soluble levels of IL-1ß protein were dependent on the inflammasome after challenge with either ozone or HA. HA challenge resulted in cleavage of macrophage-derived caspase1 and IL-1ß, suggesting a role for alveolar macrophages in Nlrp3-dependent AHR. CONCLUSIONS: The Nlrp3 inflammasome is required for the development of ozone-induced reactive airways disease.

NLRP3 inflammasome induces chemotactic immune cell migration to the CNS in experimental autoimmune encephalomyelitis.

The NLRP3 inflammasome is a multiprotein complex consisting of three kinds of proteins, NLRP3, ASC, and pro-caspase-1, and plays a role in sensing pathogens and danger signals in the innate immune system. The NLRP3 inflammasome is thought to be involved in the development of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). However, the mechanism by which the NLRP3 inflammasome induces EAE is not clear. In this study, we found that the NLRP3 inflammasome played a critical role in inducing T-helper cell migration into the CNS. To gain migratory ability, CD4(+) T cells need to be primed by NLRP3 inflammasome-sufficient antigen-presenting cells to up-regulate chemotaxis-related proteins, such as osteopontin, CCR2, and CXCR6. In the presence of the NLRP3 inflammasome, dendritic cells and macrophages also induce chemotactic ability and up-regulate chemotaxis-related proteins, such as α4ß1 integrin, CCL7, CCL8, and CXCL16. On the other hand, reduced Th17 cell population size in immunized Nlrp3(-/-) and Asc(-/-) mice is not a determinative factor for their resistance to EAE. As currently applied in clinical interventions of MS, targeting immune cell migration molecules may be an effective approach in treating MS accompanied by NLRP3 inflammasome activation.

Interferon-ß therapy against EAE is effective only when development of the disease depends on the NLRP3 inflammasome.

Interferon-ß (IFN-ß) is widely used to treat multiple sclerosis (MS), and its efficacy was demonstrated in the setting of experimental autoimmune encephalomyelitis (EAE), an animal model of MS; however, IFN-ß is not effective in treating all cases of MS. Here, we demonstrate that signaling by IFNAR (the shared receptor for IFN-α and IFN-ß) on macrophages inhibits activation of Rac1 and the generation of reactive oxygen species (ROS) through suppressor of cytokine signaling 1 (SOCS1). The inhibition of Rac1 activation and ROS generation suppressed the activity of the Nod-like receptor (NLR) family, pyrin domain-containing 3 (NLRP3) inflammasome, which resulted in attenuated EAE pathogenicity. We further found that two subsets of EAE could be defined on the basis of their dependency on the NLRP3 inflammasome and that IFN-ß was not an effective therapy when EAE was induced in an NLRP3 inflammasome-independent fashion. Thus, our study demonstrates a previously uncharacterized signaling pathway that is involved in the suppression of EAE by IFN-ß and characterizes NLRP3-independent EAE, which cannot be treated with IFN-ß.

The inflammasome regulatory pathway and infections: role in pathophysiology and clinical implications.

The innate immune system serves to generate immediate host defenses against pathogens. Advance in the mechanism of innate immunity has provided new insights into host-pathogen microbial interactions. The cytosolic multi-protein complex called the inflammasome, which regulates the caspase-1 dependent processing of inflammatory cytokines IL-1ß and IL-18, is critical for the innate defense against pathogens. We summarize the current knowledge regarding the regulatory functions of the inflammasome in the pathogenesis of infections by various microbes (e.g., bacteria, fungi, viruses, and protozoa), and discuss its potential application in a clinical setting. Understanding of the unique role of the inflammasome signaling pathway in initiating and regulating inflammation is pivotal for the development of innovative approaches to optimize management of these infections.

Surfactant protein-A inhibits mycoplasma-induced dendritic cell maturation through regulation of HMGB-1 cytokine activity.

During pulmonary infections, a careful balance between activation of protective host defense mechanisms and potentially injurious inflammatory processes must be maintained. Surfactant protein A (SP-A) is an immune modulator that increases pathogen uptake and clearance by phagocytes while minimizing lung inflammation by limiting dendritic cell (DC) and T cell activation. Recent publications have shown that SP-A binds to and is bacteriostatic for Mycoplasma pneumoniae in vitro. In vivo, SP-A aids in maintenance of airway homeostasis during M. pneumoniae pulmonary infection by preventing an overzealous proinflammatory response mediated by TNF-α. Although SP-A was shown to inhibit maturation of DCs in vitro, the consequence of DC/SP-A interactions in vivo has not been elucidated. In this article, we show that the absence of SP-A during M. pneumoniae infection leads to increased numbers of mature DCs in the lung and draining lymph nodes during the acute phase of infection and, consequently, increased numbers of activated T and B cells during the course of infection. The findings that glycyrrhizin, a specific inhibitor of extracellular high-mobility group box-1 (HMGB-1) abrogated this effect and that SP-A inhibits HMGB-1 release from immune cells suggest that SP-A inhibits M. pneumoniae-induced DC maturation by regulating HMGB-1 cytokine activity.

The role of surfactant protein A in bleomycin-induced acute lung injury.

RATIONALE: Surfactant protein A (SP-A) is a collectin family member that has multiple immunomodulatory roles in lung host defense. SP-A levels are altered in the bronchoalveolar lavage (BAL) fluid and serum of patients with acute lung injury and acute respiratory distress syndrome, suggesting the importance of SP-A in the pathogenesis of acute lung injury. OBJECTIVES: Investigate the role of SP-A in the murine model of noninfectious lung injury induced by bleomycin treatment. METHODS: Wild-type (WT) or SP-A deficient (SP-A(-/-)) mice were challenged with bleomycin, and various indices of lung injury were analyzed. MEASUREMENTS AND MAIN RESULTS: On challenge with bleomycin, SP-A(-/-) mice had a decreased survival rate as compared with WT mice. SP-A(-/-) mice had a higher degree of neutrophil-dominant cell recruitment and the expression of the inflammatory cytokines in BAL fluid than did WT mice. In addition, SP-A(-/-) mice had increased lung edema as assessed by the increased levels of intravenously injected Evans blue dye leaking into the lungs. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and active caspase-3 staining suggested the increased apoptosis in the lung sections from SP-A(-/-) mice challenged with bleomycin. SP-A also specifically reduced bleomycin-induced apoptosis in mouse lung epithelial 12 cells in vitro. Moreover, intratracheal administration of exogenous SP-A rescued the phenotype of SP-A(-/-) mice in vivo. CONCLUSIONS: These data suggest that SP-A plays important roles in modulating inflammation, apoptosis, and epithelial integrity in the lung in response to acute noninfectious challenges.

Blimp-1/PRDM1 mediates transcriptional suppression of the NLR gene NLRP12/Monarch-1.

NLR (nucleotide-binding domain, leucine-rich repeat) proteins are intracellular regulators of host defense and immunity. One NLR gene, NLRP12 (NLR family, pyrin domain containing 12)/Monarch-1, has emerged as an important inhibitor of inflammatory gene expression in human myeloid cells. This is supported by genetic analysis linking the loss of a functional NLRP12 protein to hereditary periodic fever. NLRP12 transcription is diminished by specific TLR stimulation and myeloid cell maturation, consistent with its role as a negative regulator of inflammation. The NLRP12 promoter contains a novel Blimp-1 (B lymphocyte-induced maturation protein-1)/PRDM1 (PR domain-containing 1, with ZNF domain) binding site, and Blimp-1 reduces NLRP12 promoter activity, expression, and histone 3 acetylation. Blimp-1 associates with the endogenous NLRP12 promoter in a TLR-inducible manner and mediates the down-regulation of NLRP12 expression by TLR agonists. As expected, the expression of NLRP12 and Blimp-1 is inversely correlated. Analysis of Blimp-1(-/-) murine myeloid cells provides physiologic evidence that Blimp-1 reduces NLRP12 gene expression during cell differentiation. This demonstrates a novel role for Blimp-1 in the regulation of an NLR gene.

ATP binding by monarch-1/NLRP12 is critical for its inhibitory function.

The recently discovered nucleotide binding domain-leucine rich repeat (NLR) gene family is conserved from plants to mammals, and several members are associated with human autoinflammatory or immunodeficiency disorders. This family is defined by a central nucleotide binding domain that contains the highly conserved Walker A and Walker B motifs. Although the nucleotide binding domain is a defining feature of this family, it has not been extensively studied in its purified form. In this report, we show that purified Monarch-1/NLRP12, an NLR protein that negatively regulates NF-kappaB signaling, specifically binds ATP and exhibits ATP hydrolysis activity. Intact Walker A/B motifs are required for this activity. These motifs are also required for Monarch-1 to undergo self-oligomerization, Toll-like receptor- or CD40L-activated association with NF-kappaB-inducing kinase (NIK) and interleukin-1 receptor-associated kinase 1 (IRAK-1), degradation of NIK, and inhibition of IRAK-1 phosphorylation. The stable expression of a Walker A/B mutant in THP-1 monocytes results in increased production of proinflammatory cytokines and chemokines to an extent comparable to that in cells in which Monarch-1 is silenced via short hairpin RNA. The results of this study are consistent with a model wherein ATP binding regulates the anti-inflammatory activity of Monarch-1.

Immunomodulatory roles of surfactant proteins A and D: implications in lung disease.

Surfactant, a lipoprotein complex, was originally described for its essential role in reducing surface tension at the air-liquid interface of the lung; however, it is now recognized as being a critical component in lung immune host defense. Surfactant proteins (SP)-A and -D are pattern recognition molecules of the collectin family of C-type lectins. SP-A and SP-D are part of the innate immune system and regulate the functions of other innate immune cells, such as macrophages. They also modulate the adaptive immune response by interacting with antigen-presenting cells and T cells, thereby linking innate and adaptive immunity. Emerging studies suggest that SP-A and SP-D function to modulate the immunologic environment of the lung so as to protect the host and, at the same time, modulate an overzealous inflammatory response that could potentially damage the lung and impair gas exchange. Numerous polymorphisms of SPs have been identified that may potentially possess differential functional abilities and may act via different receptors to ultimately alter the susceptibility to or severity of lung diseases.

Monarch-1 suppresses non-canonical NF-kappaB activation and p52-dependent chemokine expression in monocytes.

CATERPILLER (NOD, NBD-LRR) proteins are rapidly emerging as important mediators of innate and adaptive immunity. Among these, Monarch-1 operates as a novel attenuating factor of inflammation by suppressing inflammatory responses in activated monocytes. However, the molecular mechanisms by which Monarch-1 performs this important function are not well understood. In this report, we show that Monarch-1 inhibits CD40-mediated activation of NF-kappaB via the non-canonical pathway in human monocytes. This inhibition stems from the ability of Monarch-1 to associate with and induce proteasome-mediated degradation of NF-kappaB inducing kinase. Congruently, silencing Monarch-1 with shRNA enhances the expression of p52-dependent chemokines.

Criteria for effective design, construction, and gene knockdown by shRNA vectors.

BACKGROUND: RNA interference (RNAi) technology is a powerful methodology recently developed for the specific knockdown of targeted genes. RNAi is most commonly achieved either transiently by transfection of small interfering (si) RNA oligonucleotides, or stably using short hairpin (sh) RNA expressed from a DNA vector or virus. Much controversy has surrounded the development of rules for the design of effective siRNA oligonucleotides; and whether these rules apply to shRNA is not well characterized. RESULTS: To determine whether published algorithms for siRNA oligonucleotide design apply to shRNA, we constructed 27 shRNAs from 11 human genes expressed stably using retroviral vectors. We demonstrate an efficient method for preparing wild-type and mutant control shRNA vectors simultaneously using oligonucleotide hybrids. We show that sequencing through shRNA vectors can be problematic due to the intrinsic secondary structure of the hairpin, and we determine a strategy for effective sequencing by using a combination of modified BigDye chemistries and DNA relaxing agents. The efficacy of knockdown for the 27 shRNA vectors was evaluated against six published algorithms for siRNA oligonucleotide design. Our results show that none of the scoring algorithms can explain a significant percentage of variance in shRNA knockdown efficacy as assessed by linear regression analysis or ROC curve analysis. Application of a modification based on the stability of the 6 central bases of each shRNA provides fair-to-good predictions of knockdown efficacy for three of the algorithms. Analysis of an independent set of data from 38 shRNAs pooled from previous publications confirms these findings. CONCLUSION: The use of mixed oligonucleotide pairs provides a time and cost efficient method of producing wild type and mutant control shRNA vectors. The addition to sequencing reactions of a combination of mixed dITP/dGTP chemistries and DNA relaxing agents enables read through the intrinsic secondary structure of problematic shRNA vectors. Six published algorithms for siRNA oligonucleotide design that were tested in this study show little or no efficacy at predicting shRNA knockdown outcome. However, application of a modification based on the central shRNA stability should provide a useful improvement to the design of effective shRNA vectors.

Direct manipulation of activator protein-1 controls thymocyte proliferation in vitro.

B cell activating transcription factor (BATF) belongs to the activator protein-1 (AP-1) superfamily of basic leucine zipper transcription factors and forms heterodimers with Jun that possess minimal transcriptional activity. Mice carrying a p56(lck)HA-BATF transgene were created to observe the effects of constitutive expression of this well-characterized AP-1 inhibitor on T cell proliferation. Consistent with the role of AP-1 in promoting the proliferation of many cell types, BATF-transgenic thymocytes proliferate poorly in vitro when stimulated with anti-CD3epsilon and anti-CD28 antibodies or with Concanavalin A. However, when BATF-transgenic thymocytes were stimulated using a standard treatment of PMA and ionomycin, proliferation is normal. The responsiveness to PMA and ionomycin can be attributed to the dramatic disappearance of the hemagglutinin antigen (HA)-tagged BATF protein which is a PKC-dependent process caused by the down-regulation of the p56(lck) proximal promoter coupled with the rapid turnover of the HA-BATF protein. These studies describe conditions of T cell stimulation that negatively influence transcription of the widely used p56(lck) proximal promoter expression cassette. In addition, the unique circumstances of this regulation were exploited to demonstrate that inhibition of AP-1 activity by BATF exerts a direct, and reversible, effect on T cell proliferation in vitro.

The CATERPILLER protein monarch-1 is an antagonist of toll-like receptor-, tumor necrosis factor alpha-, and Mycobacterium tuberculosis-induced pro-inflammatory signals.

The CATERPILLER (CLR, also NOD and NLR) proteins share structural similarities with the nucleotide binding domain (NBD)-leucine-rich repeat (LRR) superfamily of plant disease-resistance (R) proteins and are emerging as important immune regulators in animals. CLR proteins contain NBD-LRR motifs and are linked to a limited number of distinct N-terminal domains including transactivation, CARD (caspase activation and recruitment), and pyrin domains (PyD). The CLR gene, Monarch-1/Pypaf7, is expressed by resting primary myeloid/monocytic cells, and its expression in these cells is reduced by Toll-like receptor (TLR) agonists tumor necrosis factor (TNF) alpha and Mycobacterium tuberculosis. Monarch-1 reduces NFkappaB activation by TLR-signaling molecules MyD88, IRAK-1 (type I interleukin-1 receptor-associated protein kinase), and TRAF6 (TNF receptor (TNFR)-associated factor) as well as TNFR signaling molecules TRAF2 and RIP1 but not the downstream NFkappaB subunit p65. This indicates that Monarch-1 is a negative regulator of both TLR and TNFR pathways. Reducing Monarch-1 expression with small interference RNA in myeloid/monocytic cells caused a dramatic increase in NFkappaB activation and cytokine expression in response to TLR2/TLR4 agonists, TNFalpha, or M. tuberculosis infection, suggesting that Monarch-1 is a negative regulator of inflammation. Because Monarch-1 is the first CLR protein that interferes with both TLR2 and TLR4 activation, the mechanism of this interference is significant. We find that Monarch-1 associates with IRAK-1 but not MyD88, resulting in the blockage of IRAK-1 hyperphosphorylation. Mutants containing the NBD-LRR or PyD-NBD also blocked IRAK-1 activation. This is the first example of a CLR protein that antagonizes inflammatory responses initiated by TLR agonists via interference with IRAK-1 activation.

The CATERPILLER family: an ancient family of immune/apoptotic proteins.

Ancient immune pathways in other species have provided clues for the discovery of important molecules in the mammalian immune system. A notable example is the discovery of Toll-like receptors based on the Toll receptors in Drosophila. In plants, a subclass of the disease resistance (R) genes is crucial for immune defense against a host of insults. This R gene subclass encodes a combined nucleotide-binding domain/leucine rich region (NBD/LRR) motif. Intriguingly, proteins with such a motif are found in mammalians, and several are also shown to be important in inflammatory and immune responses. This family, which we designated as the CATERPILLER (CARD, Transcription Enhancer, R (purine)-binding, Pyrin, Lots of Leucine Repeats) gene family while others have designated it as the NOD family, has over 20 members. They are crucial in the control of cytokines, inflammatory responses, NF-kappaB activation, and likely cell death and survival. Several prominent members including CIITA, CIAS1, and NOD2 are linked to immunologic genetic disorders that are hereditary. This indicates that these genes are ancient and important regulators of the immune system.

CATERPILLER 16.2 (CLR16.2), a novel NBD/LRR family member that negatively regulates T cell function.

The newly discovered mammalian CATERPILLER (NOD, NALP, PAN) family of proteins share similarities with the NBD-LRR superfamily of plant disease resistance (R) proteins and are predicted to mediate important immune regulatory function. This report describes the first cloning and characterization of a novel CATERPILLER gene, CLR16.2 that is located on human chromosome 16. The protein encoded by this gene has a typical NBD-LRR configuration. Analysis of CLR16.2 suggests the highest expression among T lymphocytes. Cellular localization studies of CLR16.2 revealed that it is a cytoplasmic protein. Querying microarray studies in the public data base showed that CLR16.2 was significantly (>90%) down-regulated 6 h after anti-CD3 and anti-CD28 stimulation of primary T lymphocytes. Its reduction upon T cell stimulation is consistent with a potential negative regulatory role. Indeed CLR16.2 decreased NF-kappaB, NFAT, and AP-1 induction of reporter gene constructs in response to T cell activation by anti-CD3 and anti-CD28 antibodies or PMA and ionomycin. Following T cell stimulation, the presence of CLR16.2 reduced the levels of the endogenous transcripts for the IL-2 and CD25 proteins that are central in maintaining T cell activation and preventing T cell anergy. This reduction was accompanied by a delay of IkappaBalpha degradation. We propose that CLR16.2 serves to attenuate T cell activation via TCR and co-stimulatory molecules, and its reduction during T cell stimulation allows the ensuing cellular activation.

Cutting edge: Monarch-1: a pyrin/nucleotide-binding domain/leucine-rich repeat protein that controls classical and nonclassical MHC class I genes.

Proteins containing a limited number of N-terminal motifs followed by nucleotide-binding domain and leucine-rich repeat regions are emerging as important regulators for immunity. A search of human genome scaffold databases has identified a large family of known and unknown genes, which we have recently called the CATERPILLER (caspase recruitment domain, transcription enhancer, r(purine)-binding, pyrin, lots of leucine repeats) gene family. This work describes the characterization of a new member, Monarch-1. Monarch-1 has four different splice forms due to the differential splicing of leucine-rich repeat motifs. It is expressed in cells of myeloid-monocytic origin. Affymetrix microarrays and small interfering RNA were used to elucidate the downstream effects of Monarch-1 expression in cells including those of myeloid-monocytic origin. These analyses show that Monarch-1 enhances nonclassical and classical MHC class I expression at the level of the promoter, RNA, and protein expression.

BATF transgenic mice reveal a role for activator protein-1 in NKT cell development.

The importance of regulated AP-1 activity during T cell development was assessed using transgenic mice overexpressing BATF, a basic leucine zipper transcription factor and an AP-1 inhibitor. BATF transgenic animals possess normal thymic cellularity and all major T cell subsets, but show impaired thymocyte proliferation in vitro and no induction of IL-2, IL-4, IL-5, IL-10, and IL-13 expression. Since NKT cells are largely responsible for cytokine production in the thymus, this population was examined by detection of the V alpha 14-J alpha 281 TCR, flow cytometry of NK1.1(+) TCR beta(+) cells, and analysis of cytokine production by heat-stable Ag(low) thymocytes and peripheral NKT cells stimulated in vivo. Results show a severe under-representation of NKT cells in BATF transgenic animals, providing the first evidence that the precise control of AP-1-mediated transcription is critical for the proper emergence of thymus-derived NKT cells in the mouse.