Receptor avidity and costimulation specify the intracellular Ca2+ signaling pattern in CD4(+)CD8(+) thymocytes.

Thymocyte maturation is governed by antigen-T cell receptor (TCR) affinity and the extent of TCR aggregation. Signals provided by coactivating molecules such as CD4 and CD28 also influence the fate of immature thymocytes. The mechanism by which differences in antigen-TCR avidity encode unique maturational responses of lymphocytes and the influence of coactivating molecules on these signaling processes is not fully understood. To better understand the role of a key second messenger, calcium, in governing thymocyte maturation, we measured the intracellular free calcium concentration ([Ca2+]i) response to changes in TCR avidity and costimulation. We found that TCR stimulation initiates either amplitude- or frequency-encoded [Ca2+]i changes depending on (a) the maturation state of stimulated thymocytes, (b) the avidity of TCR interactions, and (c) the participation of specific coactivating molecules. Calcium signaling within immature but not mature thymocytes could be modulated by the avidity of CD3/CD4 engagement. Low avidity interactions induced biphasic calcium responses, whereas high avidity engagement initiated oscillatory calcium changes. Notably, CD28 participation converted the calcium response to low avidity receptor engagement from a biphasic to oscillatory pattern. These data suggest that calcium plays a central role in encoding the nature of the TCR signal received by thymocytes and, consequently, a role in thymic selection.

Coupling of the TCR to integrin activation by Slap-130/Fyb.

SLAP-130/Fyb (SLP-76-associated phosphoprotein or Fyn-binding protein; also known as Fyb/Slap) is a hematopoietic-specific adapter, which associates with and modulates function of SH2-containing leukocyte phosphoprotein of 76 kilodaltons (SLP-76). T cells from mice lacking SLAP-130/Fyb show markedly impaired proliferation following CD3 engagement. In addition, the T cell receptor (TCR) in SLAP-130/Fyb mutant cells fails to enhance integrin-dependent adhesion. Although TCR-induced actin polymerization is normal, TCR-stimulated clustering of the integrin LFA-1 is defective in SLAP-130/Fyb-deficient cells. These data indicate that SLAP-130/Fyb is important for coupling TCR-mediated actin cytoskeletal rearrangement with activation of integrin function, and for T cells to respond fully to activating signals.

Bacillus Calmette-Guérin therapy alters the progression of superficial bladder cancer.

The effectiveness of BCG in preventing disease progression in patients with superficial bladder cancer is evaluated. Long-term follow-up of high-risk patients treated in a previously reported randomized control trial of intravesical plus percutaneous BCG shows that progression occurred in 41/43 (95%) of control and 23/43 (53%) of BCG-treated patients. Muscle invasive and/or metastatic disease occurred with equal frequency in the two groups, but was significantly delayed by BCG treatment (P = .012). Cystectomies were required in 18/43 (42%) control and 11/43 (26%) BCG-treated patients. Median time to cystectomy was 8 months for control v 24 months for BCG-treated patients. Based on initial treatment, survival was improved by BCG therapy (P = .032) (median follow-up 6 years). These results suggest that in high-risk patients intravesical BCG can delay disease progression, prolong the period of bladder preservation, and increase overall survival.

Chemokine signaling and HIV-1 fusion mediated by macrophage CXCR4: implications for target cell tropism.

To better understand CXCR4 function on macrophages and the relationship between coreceptor use and macrophage tropism among diverse HIV-1 isolates, we analyzed macrophage pathways involved in Env-mediated fusion, productive HIV-1 infection, and chemokine-elicited signaling. We found that both CXCR4 and CCR5 transduced intracellular signals in monocyte-derived macrophages, activating K+ and Cl- ion channels and elevating intracellular calcium in response to their chemokine ligands stromal cell-derived factor-1alpha and macrophage inflammatory protein-1beta, respectively. The prototype T-tropic X4 strain IIIB infected macrophages poorly, and this was associated with failure of the IIIB Env to fuse efficiently with target macrophages despite functional CXCR4. In contrast, several primary X4 isolates mediated efficient CXCR4-dependent fusion and productive macrophage infection. Several R5X4 strains could fuse with and infect macrophages through both CCR5 and CXCR4. Thus, macrophages express functional CXCR4 and CCR5 but primary and prototype X4 isolates differ in their ability to utilize macrophage CXCR4. Isolates classified as X4 based on coreceptor use may be phenotypically either T-tropic or dual-tropic and, conversely, phenotypically dual-tropic isolates may be either R5X4 or X4 based on coreceptor use.

Evaluation of anhydride oligomers within polymer microsphere blends and their impact on bioadhesion and drug delivery in vitro.

The effect of the addition of small molecular weight anhydride oligomers to polymer microspheres was evaluated and increased bioadhesion of the composite was demonstrated. Blends of low molecular weight anhydride oligomers with thermoplastic poly(fumaric-co-sebacic anhydride) [p(FASA)] and polycaprolactone were examined. The effects of anhydride oligomers on polymer microsphere degradation, crystallinity, and surface morphology were also explored. The results demonstrated that fumaric anhydride oligomer remained within polymer microspheres for several hours after exposure to phosphate buffer, formed a homogenous crystalline blend, increased bioadhesion as measured on rat intestine, and enhanced drug delivery in vitro as measured by the everted sac technique.

Poly(fumaric-co-sebacic anhydride). A degradation study as evaluated by FTIR, DSC, GPC and X-ray diffraction.

The degradation of three poly(fumaric-co-sebacic anhydride) [P(FA:SA)] copolymers is examined in a composition of microspheres made by the hot melt encapsulation process. The emergence of low molecular weight oligomers occurs during degradation of the copolymer microspheres, as evidenced by a variety of characterization methods. Characterization was conducted to determine the extent of degradation of the polyanhydride microspheres using Fourier-transform infrared spectroscopy (FTIR), gel permeation chromatography (GPC), differential scanning calorimetry (DSC) and X-ray diffraction. It is demonstrated that degradation of P(FA:SA) is greatly accelerated at basic pH, yet there is little difference between degradation in neutral and acidic buffers. A good correlation exists between the results of each characterization method, which allows a better understanding of the degradation process and the resulting formation of low molecular weight oligomers in poly(fumaric-co-sebacic anhydride).

Correlation of two bioadhesion assays: the everted sac technique and the CAHN microbalance.

This contribution correlates two in vitro methods utilized to determine bioadhesion. One method, the everted intestinal sac technique, is a passive test for bioadhesion involving several polymer microspheres and a section of everted intestinal tissue. The other method, the CAHN microbalance, employs a CAHN dynamic contact angle analyzer with modified software to record the tensile forces measured as a single polymer microsphere is pulled from intestinal tissue. This study demonstrates that CAHN and everted sac experiments yield similar results when used to quantify the bioadhesive nature of polymer microsphere systems. A polymer showing high adhesion in one method also demonstrates high bioadhesion in the other method; polymers that exhibit high fracture strength and tensile work measurements with the CAHN microbalance also yield high binding percentages with the everted sac method. The polymers tested and reported here are poly(caprolactone) and different copolymer ratios of poly(fumaric-co-sebacic anhydride). The results of this correlation demonstrate that each method alone is a valuable indicator of bioadhesion.

Toxoplasma gondii induces changes in intracellular calcium in macrophages.

Toxoplasma gondii is an obligate intracellular parasite that interacts with calcium storage organelles and induces calcium-dependent signalling in macrophages. This study was performed to determine whether Toxoplasma induces changes in intracellular calcium in these cells. Ratiometric imaging of live, Fura-2 loaded macrophages challenged with T. gondii revealed robust elevations in intracellular calcium. These elevations were late in onset, beginning 15-20 min after addition of parasites and occurred in up to 20% of macrophages in an imaging field. Further characterization of these events revealed that they follow from challenge with live T. gondii, but not heat-killed parasites or soluble Toxoplasma antigen (STAg). Parasite-induced calcium elevations derived from extracellular sources, and were independent of host recognition factors MyD88 and CCR5. These findings indicate that Toxoplasma gondii alters calcium homeostasis in macrophages and this activity is independent of known pathways involved in the innate recognition of this organism.

Evidence for voltage modulation of IL-2 production in mitogen-stimulated human peripheral blood lymphocytes.

The membrane potential of human PBMC was modulated in culture by isotonic high extracellular K+ (K+e), or the K+ channel blocker, charybdotoxin (ChTX), to determine the effect of depolarization on stimulated proliferation, IL-2 elaboration, and gene expression. In serum-free cultures, ChTX and high [K+]e induced a specific dose-dependent decrease in IL-2 production. ChTX inhibited proliferation of PBMC and purified T cells, decreased IL-2 elaboration 15 h after stimulation by 78.4 +/- 5.3% (n = 5), and decreased IL-2 mRNA steady-state levels by 80% between 8 and 10 h after stimulation. The IC50 for ChTX-inhibition of IL-2 elaboration and IL-2 mRNA were both 1 nM. Similarly, high [K+]e inhibited proliferation with an IC50 of 38.9 +/- 1.1 mM (n = 13), decreased IL-2 elaboration with an IC50 of 21.3 +/- 1.2 mM (n = 6), and decreased IL-2 mRNA steady-state levels with an IC50 of 18 mM. The sensitivities of both IL-2 production and proliferation to depolarization were substantially reduced by calcium, serum, and exogenous rIL-2. From these findings we conclude that membrane potential may contribute to the control of immune responsiveness in vivo.

Avian tibial dyschondroplasia. II. Biochemical changes.

Biochemical parameters (dry matter, DNA, protein, cAMP, and calmodulin) were measured in tibial dyschondroplastic (TD) cartilage. This abnormal cartilage, which is a mass of unmineralized, unvascularized cartilage found in the proximal metaphysis of the tibiotarsus and tarsometatarsus, was compared with normal epiphyseal growth plate and hypertrophic cartilage obtained from day-old embryonic cone. The latter tissue is an example of cartilage which rapidly undergoes vascularization and mineralization. When compared with normal growth plate, tibial dyschondroplastic cartilage was found to contain lower amounts of dry matter, DNA, protein, cAMP, and calmodulin. This cartilage did not respond to factors in serum which stimulate 35S uptake. Although the above two types of cartilage contained similar amounts of ash, TD cartilage had less phosphorus and potassium and more sodium than the growth plate. The two types of cartilage had similar lysozyme activity and proteoglycan (hexosamine) content. In many of the parameters measured, day-old hypertrophic cartilage was similar to the normal growth plate. However, these tissues did differ in DNA, protein, ash, and lysozyme content. Substantially greater amounts of ash and lysozyme were found in the hypertrophic cartilage, which appeared to be related to events of mineralization and vascularization of this cartilage. These events did not occur in the abnormal cartilage cells found in the tibial dyschondroplastic lesion.

A dominant negative PPARgamma mutant shows altered cofactor recruitment and inhibits adipogenesis in 3T3-L1 cells.

AIMS/HYPOTHESIS: PPARgamma, a member of the nuclear hormone receptor family of transcription factors, plays a key role in adipocyte differentiation and insulin sensitivity. The aim of this study was to identify a potential dominant negative murine PPARgamma mutant and to characterize the in vitro functional properties of this mutant. METHODS: In vitro transient transfections and mammalian two-hybrid assays in TSA201 cells were used to characterize the transcriptional activity of the L466A mutant and to study the molecular interaction of transcriptional cofactors with the L466A mutant in an attempt to elucidate the mechanism of its dominant negative activity. Adenoviral constructs expressing PPARgamma wild-type (AdWT) or the L466A mutant (AdL466A) were infected into the murine 3T3-L1 cell line to study the mutant's effect on adipogenesis. RESULTS: The L466A mutant alone is transcriptionally defective. However, it retains DNA binding and inhibits the ligand-dependent and -independent activity of the wild-type receptor, consistent with dominant negative properties. In mammalian two-hybrid studies, the L466A mutant does not bind nuclear receptor coactivators. However, it more avidly recruits corepressors due to enhanced binding to the corepressor ID1 domain, leading to pronounced transcriptional repression. The AdL466A mutant inhibits adipogenesis induced by either a differentiation cocktail or by thiazolidinedione ligand. AdL466A infection also blocked the upregulation of the adipocyte marker genes aP2 and adipsin. CONCLUSION: We conclude that the L466A PPARgamma mutant possesses potent dominant negative activity based on preferential corepressor recruitment and it inhibits adipogenesis and the expression of adipocyte-specific genes.

Identification of Kv1.1 expression by murine CD4-CD8- thymocytes. A role for voltage-dependent K+ channels in murine thymocyte development.

The patch-clamp recording technique and RNA-polymerase chain reaction were used to identify the voltage-dependent K+ channels expressed by murine fetal and adult CD4-CD8- thymocytes. Two distinct currents, encoded by the genes Kv1.1 and Kv1.3 were identified based upon their biophysical and pharmacologic characteristics and confirmed with RNA-polymerase chain reaction. Peptide blockers of Kv1.1 and Kv1.3 gene products were also applied to a murine fetal thymic organ culture system to investigate the developmental role of these K+ channels. Dendrotoxin (DTX) and charybdotoxin (CTX), antagonists of Kv1.1 and Kv1.3 channels, respectively, decreased thymocyte yields in organ culture without affecting thymocyte viability. DTX-treated thymi contained 56 +/- 8% (n = 8 experiments), and CTX-treated thymi contained 74 +/- 4% (n = 7 experiments) as many thymocytes as untreated lobes. DTX and CTX also altered the developmental progression of thymocytes in fetal organ culture. These data provide the first evidence of Kv1.1 expression in a lymphoid cell and indicate that thymocyte voltage-dependent K+ channels are critical to thymocyte preclonal expansion and/or maturation.

ATP-evoked Ca2+ transients and currents in murine thymocytes: possible role for P2X receptors in death by neglect.

The P2X family of ATP receptors (P2XR) have been implicated in thymocyte death in vitro and in vivo. We characterized ATP-evoked Ca2+ transients and membrane currents in thymocytes to better understand the role of P2XR during thymocyte development. ATP4-, but not UTP or GTP, activated a sustained non-selective cation current in voltage-clamped CD4- CD8- and CD4+ CD8+ thymocytes that was reversed by apyrase, which hydrolyzes ATP, and by the P2XR antagonists suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). The more selective P2XR agonist alphabeta-methylene ATP activated a smaller rapidly decaying current in both thymocyte populations. Reverse transcription-PCR results indicate that P2X1, P2X2, P2X6, and/or P2X7 are expressed in thymocytes. Finally, we used PPADS to examine the role of P2XR during thymocyte development in situ. PPADS-treated thymi yielded significantly more thymocytes (38%), due to a selective increase in CD4+ CD8+ cells. Together these data suggest that one or more PPADS-sensitive P2XR (P2X1, P2X2, P2X7) are involved in thymocyte apoptosis, and we propose more specifically a role associated with death by neglect.

Expression and function of voltage-dependent potassium channel genes in human airway smooth muscle.

Patch clamp and RNA-polymerase chain reaction methods were used to determine the expression of voltage-dependent potassium channel currents and mRNAs in human airway smooth muscle cells, and tension measurements were used to examine the functional role of specific potassium channel gene products in human bronchial smooth muscle. RNA from airway smooth muscle tissue revealed the presence of Kv1.2 (11 kilobases (kb)) and Kv1.5 (3.5 and 4.4 kb) transcripts, as well as Kv1.1 mRNA (9.5 kb), which has not previously been reported in smooth muscle; transcripts from other gene families were not detected. RNA-polymerase chain reaction from cultured human myocytes confirmed that the identified transcripts were expressed by smooth muscle cells. The available voltage-dependent potassium current in human airway myocytes was insensitive to charybdotoxin (200 nM) but blocked by 4-aminopyridine. Dendrotoxin (1-300 nM; inhibits Kv1.1 and Kv1.2 channels), charybdotoxin (10 nM to 1 microM; inhibits KCa and Kv1.2 channels), and glybenclamide (0.1-100 microM; inhibits KATP channels) had no effect on resting tone. Conversely, 4-aminopyridine increased resting tension with an EC50 (1.8 mM) equivalent to that observed for current inhibition (1.9 mM). Human airway myocytes express mRNA from several members of the Kv1 gene family; the channel that underlies the predominate voltage-dependent current and the regulation of basal tone appears to be Kv1.5.

Retrograde Ca2+ signaling in C2C12 skeletal myocytes in response to mitochondrial genetic and metabolic stress: a novel mode of inter-organelle crosstalk.

We have investigated the mechanism of mitochondrial-nuclear crosstalk during cellular stress in mouse C2C12 myocytes. For this purpose, we used cells with reduced mitochondrial DNA (mtDNA) contents by ethidium bromide treatment or myocytes treated with known mitochondrial metabolic inhibitors, including carbonyl cyanide m-chlorophenylhydrazone (CCCP), antimycin, valinomycin and azide. Both genetic and metabolic stresses similarly affected mitochondrial membrane potential (Deltapsim) and electron transport-coupled ATP synthesis, which was also accompanied by an elevated steady-state cytosolic Ca2+ level ([Ca2+]i). The mitochondrial stress resulted in: (i) an enhanced expression of the sarcoplasmic reticular ryanodine receptor-1 (RyR-1), hence potentiating the Ca2+ release in response to its modulator, caffeine; (ii) enhanced levels of Ca2+-responsive factors calineurin, calcineurin-dependent NFATc (cytosolic counterpart of activated T-cell-specific nuclear factor) and c-Jun N-terminal kinase (JNK)-dependent ATF2 (activated transcription factor 2); (iii) reduced levels of transcription factor, NF-kappaB; and (iv) enhanced transcription of cytochrome oxidase Vb (COX Vb) subunit gene. These cellular changes, including the steady-state [Ca2+]i were normalized in genetically reverted cells which contain near-normal mtDNA levels. We propose that the mitochondria-to-nucleus stress signaling occurs through cytosolic [Ca2+]i changes, which are likely to be due to reduced ATP and Ca2+ efflux. Our results indicate that the mitochondrial stress signal affects a variety of cellular processes, in addition to mitochondrial membrane biogenesis.

HIV-1 gp120 and chemokine activation of Pyk2 and mitogen-activated protein kinases in primary macrophages mediated by calcium-dependent, pertussis toxin-insensitive chemokine receptor signaling.

Human immunodeficiency virus type 1 (HIV-1) uses the chemokine receptors CCR5 and CXCR4 as coreceptors for entry. It was recently demonstrated that HIV-1 glycoprotein 120 (gp120) elevated calcium and activated several ionic signaling responses in primary human macrophages, which are important targets for HIV-1 in vivo. This study shows that chemokine receptor engagement by both CCR5-dependent (R5) and CXCR4-dependent (X4) gp120 led to rapid phosphorylation of the focal adhesion-related tyrosine kinase Pyk2 in macrophages. Pyk2 phosphorylation was also induced by macrophage inflammatory protein-1beta (MIP-1beta) and stromal cell-derived factor-1alpha, chemokine ligands for CCR5 and CXCR4. Activation was blocked by EGTA and by a potent blocker of calcium release-activated Ca++ (CRAC) channels, but was insensitive to pertussis toxin (PTX), implicating CRAC-mediated extracellular Ca++ influx but not Galpha(i) protein-dependent mechanisms. Coreceptor engagement by gp120 and chemokines also activated 2 members of the mitogen-activated protein kinase (MAPK) superfamily, c-Jun amino-terminal kinase/stress-activated protein kinase and p38 MAPK. Furthermore, gp120-stimulated macrophages secreted the chemokines monocyte chemotactic protein-1 and MIP-1beta in a manner that was dependent on MAPK activation. Thus, the gp120 signaling cascade in macrophages includes coreceptor binding, PTX-insensitive signal transduction, ionic signaling including Ca++ influx, and activation of Pyk2 and MAPK pathways, and leads to secretion of inflammatory mediators. HIV-1 Env signaling through these pathways may contribute to dysregulation of uninfected macrophage functions, new target cell recruitment, or modulation of macrophage infection.

HIV-1 gp120 and chemokines activate ion channels in primary macrophages through CCR5 and CXCR4 stimulation.

HIV type 1 (HIV-1) uses the chemokine receptors CCR5 and CXCR4 as coreceptors for entry into target cells. Here we show that the HIV-1 envelope gp120 (Env) activates multiple ionic signaling responses in primary human macrophages, which are important targets for HIV-1 in vivo. Env from both CCR5-dependent JRFL (R5) and CXCR4-dependent IIIB (X4) HIV-1 opened calcium-activated potassium (K(Ca)), chloride, and calcium-permeant nonselective cation channels in macrophages. These signals were mediated by CCR5 and CXCR4 because macrophages lacking CCR5 failed to respond to JRFL and an inhibitor of CXCR4 blocked ion current activation by IIIB. MIP-1beta and SDF-1alpha, chemokine ligands for CCR5 and CXCR4, respectively, also activated K(Ca) and Cl(-) currents in macrophages, but nonselective cation channel activation was unique to gp120. Intracellular Ca(2+) levels were also elevated by gp120. The patterns of activation mediated by CCR5 and CXCR4 were qualitatively similar but quantitatively distinct, as R5 Env activated the K(Ca) current more frequently, elicited Cl(-) currents that were approximately 2-fold greater in amplitude, and elevated intracellular Ca(+2) to higher peak and steady-state levels. Env from R5 and X4 primary isolates evoked similar current responses as the corresponding prototype strains. Thus, the interaction of HIV-1 gp120 with CCR5 or CXCR4 evokes complex and distinct signaling responses in primary macrophages, and gp120-evoked signals differ from those activated by the coreceptors' chemokine ligands. Intracellular signaling responses of macrophages to HIV-1 may modulate postentry steps of infection and cell functions apart from infection.

Differential requirement for SLP-76 domains in T cell development and function.

The hematopoietic cell-specific adaptor protein, SLP-76, is critical for T cell development and mature T cell receptor (TCR) signaling; however, the structural requirements of SLP-76 for mediating thymopoiesis and mature T cell function remain largely unknown. In this study, transgenic mice were generated to examine the requirements for specific domains of SLP-76 in thymocytes and peripheral T cells in vivo. Examination of mice expressing various mutants of SLP-76 on the null background demonstrates a differential requirement for specific domains of SLP-76 in thymocytes and T cells and provides new insight into the molecular mechanisms underlying SLP-76 function.