Agonist-selected T cell development requires strong T cell receptor signaling and store-operated calcium entry.

T cell receptor (TCR) signaling driven by interaction of the TCR with specific complexes of self-peptide and the major histocompatibility complex determines T cell fate in thymic development. However, the signaling pathway through which TCR signal strength regulates distinct T cell lineages remains unknown. Here we have used mice lacking the endoplasmic reticulum Ca2+ sensors stromal interaction molecule 1 (STIM1) and STIM2 to show that STIM-induced store-operated Ca2+ entry is not essential for thymic development of conventional TCRαß+ T cells but is specifically required for the development of agonist-selected T cells (regulatory T cells, invariant natural killer T cells, and TCRαß+ CD8αα+ intestinal intraepithelial lymphocytes). The severe impairment of agonist-selected T cell development is mainly due to a defect in interleukin-2 (IL-2) or IL-15 signaling. Thus, STIM1 and STIM2-mediated store-operated Ca2+ influx, leading to efficient activation of NFAT (nuclear factor of activated T cells), is critical for the postselection maturation of agonist-selected T cells.

Enhanced Reactive Oxygen Species Production, Acidic Cytosolic pH and Upregulated Na+/H+ Exchanger (NHE) in Dicer Deficient CD4+ T Cells.

BACKGROUND: MicroRNAs (miRNAs) negatively regulate gene expression at a post-transcriptional level. Dicer, a cytoplasmic RNase III enzyme, is required for the maturation of miRNAs from precursor miRNAs. Dicer, therefore, is a critical enzyme involved in the biogenesis and processing of miRNAs. Several biological processes are controlled by miRNAs, including the regulation of T cell development and function. T cells generate reactive oxygen species (ROS) with parallel H+ extrusion accomplished by the Na+/H+-exchanger 1 (NHE1). The present study explored whether ROS production, as well as NHE1 expression and function are sensitive to the lack of Dicer (miRNAs deficient) and could be modified by individual miRNAs. METHODS: CD4+ T cells were isolated from CD4 specific Dicer deficient (DicerΔ/Δ) mice and the respective control mice (Dicerfl/fl). Transcript and protein levels were quantified with RT-PCR and Western blotting, respectively. For determination of intracellular pH (pHi) cells were incubated with the pH sensitive dye bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) and Na+/H+ exchanger (NHE) activity was calculated from re-alkalinization after an ammonium pulse. Changes in cell volume were measured using the forward scatter in flow cytometry, and ROS production utilizing 2',7' -dichlorofluorescin diacetate (DCFDA) fluorescence. Transfection of miRNA-control and mimics in T cells was performed using DharmaFECT3 reagent. RESULTS: ROS production, cytosolic H+ concentration, NHE1 transcript and protein levels, NHE activity, and cell volume were all significantly higher in CD4+ T cells from DicerΔ/Δ mice than in CD4+ T cells from Dicerfl/fl mice. Furthermore, individual miR-200b and miR-15b modify pHi and NHE activity in Dicerfl/fl and DicerΔ/Δ CD4+ T cells, respectively. CONCLUSIONS: Lack of Dicer leads to oxidative stress, cytosolic acidification, upregulated NHE1 expression and activity as well as swelling of CD4+ T cells, functions all reversed by miR-15b or miR-200b.

Chloride ion transport and overexpression of TMEM16A in a guinea-pig asthma model.

BACKGROUND: TMEM16A, a Ca-activated Cl channel, regulates various physiological functions such as mucin secretion. However, the role of TMEM16A in hyper-secretion in asthma is not fully understood. OBJECTIVE: The aim of this study is to evaluate Cl ion transport via TMEM16A and determine the localization of TMEM16A in a guinea-pig asthma model. METHODS: Guinea-pigs were sensitized with ovalbumin (OVA) i.p. on Days 1 and 8. On Day 22, we assessed OVA challenge-induced Cl ion transport in the sensitized tracheas ex vivo in an Ussing chamber, compared with the non-sensitized tracheas. We then examined the effect of T16Ainh-A01, a TMEM16A inhibitor, on the increase in Cl ion transport. The tracheal epithelium was immunostained with an anti-TMEM16A antibody. Epithelial cells from guinea-pig tracheas were cultured at the air-liquid interface in the presence of IL-13 for in vitro study. We studied the effect of TMEM16A inhibitors on Ca-dependent agonist, uridine triphosphate (UTP)-induced increases in Cl ion transport in the cultured cells. The cells were immunostained with an anti-TMEM16A antibody, an anti-MUC5AC antibody and an anti-α-tubulin antibody. RESULTS: OVA challenge induced an increase in short circuit current within 1 min in the OVA-sensitized tracheas but not in the non-sensitized tracheas, which was inhibited by pretreatment of T16Ainh-A01. Sensitized tracheas showed goblet cell metaplasia with more positive TMEM16A immunostaining, particularly in the apical portion compared with the non-sensitized tracheas. The in vitro UTP-induced increase in Cl ion transport was strongly inhibited by pretreatment with T16Ainh-A01, benzbromarone, and niflumic acid. TMEM16A was positively immunostained at the apical portion and in the MUC5AC-positive area in IL-13-induced goblet cell metaplasia. CONCLUSIONS: Antigen challenge and Ca-dependent agonist treatment increased Cl ion transport via the overexpression of TMEM16A in goblet cell metaplasia in a guinea-pig asthma model. TMEM16A inhibitors may be useful for the treatment of hyper-secretion in asthma.

Potassium efflux fires the canon: Potassium efflux as a common trigger for canonical and noncanonical NLRP3 pathways.

Murine caspase-11 and its human orthologues, caspase-4 and caspase-5, activate an inflammatory response following cytoplasmic recognition of cell wall constituents from Gram-negative bacteria, such as LPS. This inflammatory response involves pyroptotic cell death and the concomitant release of IL-1α, as well as the production of IL-1ß and IL-18 through the noncanonical NLR family, pyrin domain containing 3 (NLRP3) pathway. This commentary discusses three papers in this issue of the European Journal of Immunology that advance our understanding of the roles of caspase-11, -4, and -5 in the noncanonical pathway. By utilizing the new gene editing technique, clustered regularly interspaced short palindromic repeats (CRISPR), as well as sensitive cell imaging techniques, these papers establish that cytoplasmic LPS-dependent IL-1ß production requires the NLRP3 inflammasome and that its activation is dependent on K(+) efflux, whereas IL-1α release and pyroptotic cell death pathways are NLRP3-independent. These findings expand on previous research implicating K(+) efflux as the principal trigger for NLRP3 activation and suggest that canonical and noncanonical NLRP3 pathways are not as dissimilar as first thought.

Caspase-11 activates a canonical NLRP3 inflammasome by promoting K(+) efflux.

Recognition of microbe-associated molecular patterns or endogenous danger signals by a subset of cytosolic PRRs results in the assembly of multiprotein signaling complexes, the so-called inflammasomes. Canonical inflammasomes are assembled by NOD-like receptor (NLR) or PYHIN family members and activate caspase-1, which promotes the induction of pyroptosis and the release of mature interleukin-1ß/-18. Recently, a noncanonical inflammasome pathway was discovered that results in caspase-11 activation in response to bacterial lipopolysaccharide (LPS) in the cytosol. Interestingly, caspase-11 induces pyroptosis by itself, but requires NLRP3, the inflammasome adapter ASC, and caspase-1 to promote cytokine secretion. Here, we have studied the mechanism by which caspase-11 controls IL-1ß secretion. Investigating NLRP3/ASC complex formation, we find that caspase-11 functions upstream of a canonical NLRP3 inflammasome. The activation of NLRP3 by caspase-11 during LPS transfection is a cell-intrinsic process and is independent of the release of danger signals. Furthermore, we show that active caspase-11 leads to a drop of intracellular potassium levels, which is necessary to activate NLRP3. Our study, therefore, sheds new light on the mechanism of noncanonical inflammasome signaling.

Zinc piracy as a mechanism of Neisseria meningitidis for evasion of nutritional immunity.

The outer membrane of Gram-negative bacteria functions as a permeability barrier that protects these bacteria against harmful compounds in the environment. Most nutrients pass the outer membrane by passive diffusion via pore-forming proteins known as porins. However, diffusion can only satisfy the growth requirements if the extracellular concentration of the nutrients is high. In the vertebrate host, the sequestration of essential nutrient metals is an important defense mechanism that limits the growth of invading pathogens, a process known as "nutritional immunity." The acquisition of scarce nutrients from the environment is mediated by receptors in the outer membrane in an energy-requiring process. Most characterized receptors are involved in the acquisition of iron. In this study, we characterized a hitherto unknown receptor from Neisseria meningitidis, a causative agent of sepsis and meningitis. Expression of this receptor, designated CbpA, is induced when the bacteria are grown under zinc limitation. We demonstrate that CbpA functions as a receptor for calprotectin, a protein that is massively produced by neutrophils and other cells and that has been shown to limit bacterial growth by chelating Zn²âº and Mn²âº ions. Expression of CbpA enables N. meningitidis to survive and propagate in the presence of calprotectin and to use calprotectin as a zinc source. Besides CbpA, also the TonB protein, which couples energy of the proton gradient across the inner membrane to receptor-mediated transport across the outer membrane, is required for the process. CbpA was found to be expressed in all N. meningitidis strains examined, consistent with a vital role for the protein when the bacteria reside in the host. Together, our results demonstrate that N. meningitidis is able to subvert an important defense mechanism of the human host and to utilize calprotectin to promote its growth.

Anergic responses characterize a large fraction of human autoreactive naive B cells expressing low levels of surface IgM.

B cell anergy represents an important mechanism of peripheral immunological tolerance for mature autoreactive B cells that escape central tolerance enforced by receptor editing and clonal deletion. Although well documented in mice, the extent of its participation in human B cell tolerance remains to be fully established. In this study, we characterize the functional behavior of strictly defined human naive B cells separated on the basis of their surface IgM (sIgM) expression levels. We demonstrate that cells with lower sIgM levels (IgM(lo)) are impaired in their ability to flux calcium in response to either anti-IgM or anti-IgD cross-linking and contain a significantly increased frequency of autoreactive cells compared with naive B cells with higher levels of sIgM. Phenotypically, in healthy subjects, IgM(lo) cells are characterized by the absence of activation markers, reduction of costimulatory molecules (CD19 and CD21), and increased levels of inhibitory CD22. Functionally, IgM(lo) cells display significantly weaker proliferation, impaired differentiation, and poor Ab production. In aggregate, the data indicate that hyporesponsiveness to BCR cross-linking associated with sIgM downregulation is present in a much larger fraction of all human naive B cells than previously reported and is likely to reflect a state of anergy induced by chronic autoantigen stimulation. Finally, our results indicate that in systemic lupus erythematosus patients, naive IgM(lo) cells display increased levels of CD95 and decreased levels of CD22, a phenotype consistent with enhanced activation of autoreactive naive B cells in this autoimmune disease.

T cell receptor-initiated calcium release is uncoupled from capacitative calcium entry in Itk-deficient T cells.

Itk, a Tec family tyrosine kinase, plays an important but as yet undefined role in T cell receptor (TCR) signaling. Here we show that T cells from Itk-deficient mice have a TCR-proximal signaling defect, resulting in defective interleukin 2 secretion. Upon TCR stimulation, Itk-/- T cells release normal amounts of calcium from intracellular stores, but fail to open plasma membrane calcium channels. Since thapsigargin-induced store depletion triggers normal calcium entry in Itk-/- T cells, an impaired biochemical link between store depletion and channel opening is unlikely to be responsible for this defect. Biochemical studies indicate that TCR-induced inositol 1,4,5 tris-phosphate (IP3) generation and phospholipase C gamma1 tyrosine phosphorylation are substantially reduced in Itk-/- T cells. In contrast, TCR-zeta and ZAP-70 are phosphorylated normally, suggesting that Itk functions downstream of, or in parallel to, ZAP-70 to facilitate TCR-induced IP3 production. These findings support a model in which quantitative differences in cytosolic IP3 trigger distinct responses, and in which only high concentrations of IP3 trigger the influx of extracellular calcium.

TfR1 Extensively Regulates the Expression of Genes Associated with Ion Transport and Immunity.

Transferrin receptor 1 (TfR1), encoded by the TFRC gene, is the gatekeeper of cellular iron uptake for cells. A variety of molecular mechanisms are at work to tightly regulate TfR1 expression, and abnormal TfR1 expression has been associated with various diseases. In the current study, to determine the regulation pattern of TfR1, we cloned and overexpressed the human TFRC gene in HeLa cells. RNA-sequencing (RNA-seq) was used to analyze the global transcript levels in overexpressed (OE) and normal control (NC) samples. A total of 1669 differentially expressed genes (DEGs) were identified between OE and NC. Gene ontology (GO) analysis was carried out to explore the functions of the DEGs. It was found that multiple DEGs were associated with ion transport and immunity. Moreover, the regulatory network was constructed on basis of DEGs associated with ion transport and immunity, highlighting that TFRC was the node gene of the network. These results together suggested that precisely controlled TfR1 expression might be not only essential for iron homeostasis, but also globally important for cell physiology, including ion transport and immunity.

Myeloid CD11c+ Antigen-Presenting Cells Ablation Prevents Hypertension in Response to Angiotensin II Plus High-Salt Diet.

Increasing evidence shows that antigen-presenting cells (APCs) are involved in the development of inflammation associated to hypertension. However, the potential role of APCs in the modulation of renal sodium transport has not been addressed. We hypothesized that APCs participate in renal sodium transport and, thus, development of high blood pressure in response to angiotensin II plus a high-salt diet. Using transgenic mice that allow the ablation of CD11chigh APCs, we studied renal sodium transport, the intrarenal renin-angiotensin system components, blood pressure, and cardiac/renal tissue damage in response to angiotensin II plus a high-salt diet. Strikingly, we found that APCs are required for the development of hypertension and that the ablation/restitution of APCs produces rapid changes in the blood pressure in mice with angiotensin II plus a high-salt diet. Moreover, APCs were necessary for the induction of intrarenal renin-angiotensin system components and affected the modulation of natriuresis and tubular sodium transporters. Consistent with the prevention of hypertension, the ablation of APCs also prevented cardiac hypertrophy and the induction of several indicators of renal and cardiac damage. Thus, our findings indicate a prominent role of APCs as modulators of blood pressure by mechanisms including renal sodium handling, with kinetics that suggest the involvement of tubular cell functions in addition to the modulation of inflammation and adaptive immune response.

Activation of human eosinophils via P2 receptors: novel findings and future perspectives.

A growing body of information indicates that release of intracellular nucleotides represents an important way to modulate several cell pathways in physiological or pathological conditions. Nucleotides released as a consequence of cell damage, cell stress, bacterial infection, or other noxious stimuli signal at a class of plasma membrane receptors--P2 receptors--activating diverse intracellular pathways in many tissues and organs. For example, nucleotides secreted in the airway system control chloride/liquid secretion, goblet cell degranulation, and ciliary beat frequency. Several studies indicate that nucleotides play a role in airway diseases through their action on multiple cell types, including mast cells, dendritic cells, neurons, and eosinophils. Recent work by us and other groups led to the identification and characterization of P2 receptors expressed by human eosinophils. In this review, we will summarize recent developments in this field and put forward a hypothesis about the role of P2 receptors in pathophysiological conditions where eosinophils are major players.

The roles of ribosomal protein S19 C-terminus in a shortened neutrophil lifespan through delta lactoferrin.

Cell lifespan is partially regulated by a balance between survival signals via constitutively active G protein-coupled receptors (GPCRs) and death signals via death receptors. We have demonstrated that neutrophils produce a mimic ligand of G protein-coupled C5a receptor (C5aR), ribosomal protein S19 (RP S19) polymer. In contrast to an original ligand C5a, RP S19 polymer induces not only inhibition of the guanine nucleotide exchange factor activity but also initiation of the regulator of G protein signaling 3 (RGS3) promoter in a RP S19 C-terminus dependent manner. To examine an antagonistic effect of the RP S19 C-terminus on G proteins, His-S-tagged C5a or C5a/RP S19, in which an RP S19 C-terminus is bound to the C5a C-terminus, was incubated with neutrophils, and a transcription factor delta lactoferrin (δLf) was identified as a specific binding protein via pull-down experiments. The S-tagged C5a-induced agonistic effects on chemotaxis, cytoplasmic Ca(2+) influx and p38 mitogen-activated protein kinase phosphorylation were not changed by Lf knockdown and δLf overexpression in neutrophil-like or macrophage-like cells, which were differentiated into mature cells from human promyelocytic leukemia HL-60 cells by dimethyl sulfoxide and phorbol-12-myristate-13-acetate, respectively. While, the S-tagged C5a/RP S19-induced antagonistic or agonistic effects on mature HL-60 neutrophil-like or macrophage-like cells were reversed by Lf knockdown and δLf overexpression, respectively. Moreover, RGS3 expression was increased in another HL-60 neutrophil-like cells under spontaneous apoptosis induced by an apoptotic inducer MnCl2. The RGS3 expression in apoptotic neutrophil-like cells was delayed not only by Lf knockdown but also by neutralization of the RP S19 polymer or C5aR. The inhibitory extension from G protein of C5aR to Gα subsets of constitutively active GPCRs along with the RP S19 polymer-induced translocation of δLf from the cytoplasmic face of the plasma membrane to the nucleus seems to shorten the neutrophil cell lifespan.

Intracellular ionic changes induced by bullous pemphigoid IgG subclasses.

To ascertain whether membrane signal transduction is induced by bullous pemphigoid (BP) antibody and whether cell lysis is induced by its complement activation, we assessed the intracellular Ca2+ concentration ([Ca2+]i), intracellular pH, membrane potential and morphology of living cells by following the time course of fluorescence intensity of Fluo-3/AM, Snaff-1/AM, Dioc-5 and Luciffer yellow, respectively. A transient increase of Fluo-3 fluorescence intensity in DJM-1 cells (a squamous cell carcinoma line) was revealed when the cells were incubated with 2 of five IgG1 BP antibodies. However, no transient increase of Fluo-3 fluorescence intensity was revealed when the cells were incubated with IgG2 and IgG4 BP antibodies. A transient increase of Fluo-3 fluorescence intensity was revealed in DJM-1 cells incubated with 3 of seven IgG1 and 1 of four IgG2 BP antibodies in an EGTA-containing low-Ca2+ medium. On the other hand, the Dioc-5 fluorescence intensity did not change significantly, though the increase of Fluo-3 fluorescence intensity was observed. The increase of Snarf-1 fluorescence intensity was revealed in DJM-1 cells incubated with 2 of five IgG1 BP antibodies, but was not revealed in the cells incubated with IgG2 or IgG4 of BP antibodies. Study of complement activation by BP IgG1 showed a transient increase of Fluo-3 fluorescence intensity of with 3 of five IgG1 BP antibodies when DJM-1 cells were incubated with complement-supplemented normal-Ca2+ medium. At the same time, however, endocytosis and cell lysis were not observed with 2 IgG1 BP antibodies which did induce an increase of Fluo-3 fluorescence intensity when Lucifer-yellow-loaded DJM-1 cells were incubated with complement-supplemented normal-Ca2+ medium. We examined next whether anti-180 kD BP antigen monoclonal antibodies (mAbs R-223 and 233) induce an increase of Fluo-3 fluorescence intensity. MAb R-223 did not induce any increase of Fluo-3 fluorescence intensity in DJM-1 cells, when incubated with normal- and low-Ca2+ media However, mAb R-223 induced a transient increase of Fluo-3 fluorescence intensity in DJM-1 cells when incubated with complement-supplemented normal-Ca2+ medium. MAb 233 did not induced an increase of Fluo-3 fluorescence intensity in DJM-1 cells when incubated with normal- and low-Ca2+ media. These results suggest that the BP IgG1 induces Ca2+ release from intracellular storage sites, however, the complement activated by BP IgG1 does not induce cell lysis. It could not be confirmed that anti-180 kD BP antigen antibody induced Ca2+ release from intracellular storage sites.

Cystic fibrosis transmembrane conductance regulator recruitment to phagosomes in neutrophils.

Optimal microbicidal activity of human polymorphonuclear leukocytes (PMN) relies on the generation of toxic agents such as hypochlorous acid (HOCl) in phagosomes. HOCl formation requires H2O2 produced by the NADPH oxidase, myeloperoxidase derived from azurophilic granules, and chloride ion. Chloride transport from cytoplasm into phagosomes requires chloride channels which include cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel. However, the phagosomal targeting of CFTR in PMN has not been defined. Using human peripheral blood PMN, we determined that 95-99% of lysosomal-associated membrane protein 1 (LAMP-1)-positive mature phagosomes were CFTR positive, as judged by immunostaining and flow cytometric analysis. To establish a model cell system to evaluate CFTR phagosomal recruitment, we stably expressed enhanced green fluorescent protein (EGFP) alone, EGFP-wt-CFTR and EGFP-DF508-CFTR fusion proteins in promyelocytic PLB-985 cells, respectively. After differentiation into neutrophil-like cells, CFTR presentation to phagosomes was examined. EGFP-wt-CFTR was observed to associate with phagosomes and colocalize with LAMP-1. Flow cytometric analysis of the isolated phagosomes indicated that such a phagosomal targeting was determined by the CFTR portion of the fusion protein. In contrast, significantly less EGFP-DF508-CFTR was found in phagosomes, indicating a defective targeting of the molecule to the organelle. Importantly, the CFTR corrector compound VRT-325 facilitated the recruitment of DF508-CFTR to phagosomes. These data demonstrate the possibility of pharmacologic correction of impaired recruitment of mutant CFTR, thereby providing a potential means to augment chloride supply to the phagosomes of PMN in patients with cystic fibrosis to enhance their microbicidal function.

Effects of T cell-induced colonic inflammation on epithelial barrier function.

BACKGROUND: Epithelial barrier disturbance is thought to contribute to the pathogenesis of inflammatory bowel diseases; however, it remains unclear whether it is a primary defect participating to the onset of inflammation or only a consequence of sustained inflammation. METHODS: A time course study of epithelial barrier functions and immune mediators was performed in the CD4(+)CD45RB(hi) T cell transfer model of colitis using Ussing chambers. RESULTS: In nonreconstituted severe combined immunodeficiency (SCID) mice, no epithelial dysfunction was observed. However, after transfer of CD4(+)CD45RB(hi) T cells or total CD4(+) T cells, colon of SCID mice displayed a decreased epithelial resistance, even before overt microscopic inflammation had occurred. Sustained colitis of CD4(+)CD45RB(hi) T cell reconstituted mice was also associated with enhanced subepithelial resistance, enhanced paracellular permeability, and decreased net ion transport. All these reflect a disturbance of barrier function and may contribute to diarrhea. Epithelial resistance was positively correlated with interleukin 10 (IL-10) and transforming growth factor beta (TGF-beta) levels and net ion transport inversely correlated with tumor necrosis factor alpha (TNF-alpha) levels, pointing to the protective effect of IL-10 and TGF-beta and to a damaging effect of TNF-alpha. Indomethacin, a nonselective COX inhibitor, decreased epithelial resistance independent of T cells and inflammation, but its effect was more pronounced in inflamed colon. CONCLUSIONS: Induction of colitis by transfer of CD4(+)CD45RB(hi) T cells in SCID mice leads to changes in the colonic epithelium before colitis develops. Decreased epithelium resistance might contribute to the development of colitis; however, it is not sufficient to lead to chronic inflammation.

Iron in innate immunity: starve the invaders.

Iron is essential for nearly all living organisms. Innate immunity effectively restricts iron availability to microbial invaders. Some microbes have evolved effective countermeasures that blunt the effect of iron restriction. Recent epidemiologic studies have highlighted the potentiating effect of iron on microbial infections. Laboratory studies have focused on specific immune mechanisms that mediate iron withholding from microbes constitutively and in response to infections. Specialized inflammation-regulated proteins chelate iron, trap siderophores, and transport iron or modulate its transport to alter its tissue distribution during infections.

Effects of neuropeptide Y and substance P on antigen-induced ion secretion in rat jejunum.

We previously described a model of intestinal hypersensitivity in which isolated gut segments from sensitized rats demonstrated a rapid epithelial secretory response to luminal antigen that was mediated by mucosal mast cells and capsaicin-sensitive nerves. In this study, we examined the ability of the inhibitory neuropeptide, neuropeptide Y (NPY), to diminish the antigen-induced secretory response. Rats were sensitized to egg albumin (EA), and 12-14 days later, jejunal tissue was excised and mounted in Ussing chambers. NPY inhibited the short-circuit current (Isc) increase and Cl- secretion evoked by addition of EA to the luminal side of the tissue; neural blockade with tetrodotoxin (TTX) had a similar inhibitory effect. In contrast, NPY was much less effective, and TTX was completely ineffective, on the response to serosal antigen. Additional experiments examined the cell target for NPY action. NPY and TTX almost abolished the Isc response to electrical transmural stimulation of enteric nerves, suggesting a possible neural site of action. In addition, NPY significantly reduced baseline Isc; this inhibition involved both TTX-dependent and TTX-independent components. Because nerves were previously shown to facilitate antigen uptake and substance P was implicated in the response to only luminal antigen, we postulated that NPY was inhibiting nerves that facilitate antigen transport from the lumen to effector cells in the lamina propria. We therefore examined the effect of exogenous substance P added after NPY inhibition. Substance P restored the luminal antigen-induced secretory response to pretreatment values. We conclude that the neuropeptides play a significant role in immunophysiology by acting at neural and epithelial sites in the intestinal mucosa.

Visualizing the dynamics of T cell activation: intracellular adhesion molecule 1 migrates rapidly to the T cell/B cell interface and acts to sustain calcium levels.

T cell recognition typically involves both the engagement of a specific T cell receptor with a peptide/major histocompatibility complex (MHC) and a number of accessory interactions. One of the most important interactions is between the integrin lymphocyte function-associated antigen 1 (LFA-1) on the T cell and intracellular adhesion molecule 1 (ICAM-1) on an antigen-presenting cell. By using fluorescence video microscopy and an ICAM-1 fused to a green fluorescent protein, we find that the elevation of intracellular calcium in the T cell that is characteristic of activation is followed almost immediately by the rapid accumulation of ICAM-1 on a B cell at a tight interface between the two cells. This increased density of ICAM-1 correlates with the sustained elevation of intracellular calcium in the T cell, known to be critical for activation. The use of peptide/MHC complexes and ICAM-1 on a supported lipid bilayer to stimulate T cells also indicates a major role for ICAM-1/LFA-1 in T cell activation but, surprisingly, not for adhesion, as even in the absence of ICAM-1 the morphological changes and adhesive characteristics of an activated T cell are seen in this system. We suggest that T cell antigen receptor-mediated recognition of a very small number of MHC/peptide complexes could trigger LFA-1/ICAM-1 clustering and avidity regulation, thus amplifying and stabilizing the production of second messengers.

Role of calcium influx in cytotoxic T lymphocyte lytic granule exocytosis during target cell killing.

One mechanism cytotoxic T lymphocytes use to kill targets is exocytosis of cytotoxic agents from lytic granules, a process that requires Ca(2+) influx. We investigated the role of Ca(2+) influx in granule exocytosis using TALL-104 human leukemic cytotoxic T cells triggered via a bispecific antibody containing an anti-CD3 F(ab') to kill Raji B lymphoma cells. Using a novel fluorescence method, we detected target-directed release of approximately 15% of lytic granules during killing. Consistent with previous work, we observed sustained CTL Ca(2+) gradients during killing, but gradients reflect the behavior of Fura-2 in granules. Rapid imaging experiments suggest that Ca(2+) channels are not polarized during killing, indicating that Ca(2+) influx does not direct granule reorientation. Furthermore, we find that Ca(2+) acts via a high-affinity interaction to promote granule exocytosis.

Calcium efflux and influx in f-met-leu-phe (fMLP)-activated human neutrophils are chronologically distinct events.

The kinetics of efflux of calcium mobilized from intracellular stores following activation of human neutrophils with the synthetic chemotactic tripeptide, fMLP (1 microM), as well as that of the subsequent store-operated influx of this cation, has been measured by radiometric procedures using 45Ca. These procedures enabled distinction between net efflux and influx of 45Ca. Preincubation of neutrophils in medium containing 45Ca as the sole source of Ca2+, followed by activation with fMLP, resulted in a rapid efflux of the cation, which coincided with its release from intracellular stores. Efflux terminated at approximately 30 s after addition of fMLP to neutrophils and resulted in the loss of 42 +/- 3% (P < 0.005) of cell-associated 45Ca. Net influx of 45Ca, which was insensitive to the voltage-dependent Ca2+ channel blockading agent, verapamil (20 microM), could only be detected at 30-60 s after the addition of fMLP to neutrophils, and proceeded for about 5 min, resulting in intracellular concentrations of Ca2+ which were 27 +/- 3% (P<0.05) higher than preactivation levels. These results demonstrate that the efflux of cytoplasmic Ca2+ mobilized from intracellular stores during activation of neutrophils by fMLP, and the subsequent influx of extracellular Ca2+ to replete these stores, are chronologically distinct events in fMLP-activated neutrophils.