A single early activation of invariant NK T cells confers long-term protection against collagen-induced arthritis in a ligand-specific manner

The glycosphingolipid alpha-galactosylceramide (alpha-GalCer) has been shown to be a potent activator of invariant NKT (iNKT) cells, rapidly inducing large amounts of both Th1 and Th2 cytokines upon injection in mice. The C-glycoside analog of alpha-GalCer (alpha-C-GalCer), by contrast, results in an enhanced Th1-type response upon activation of iNKT cells. We administered a single dose of these Ags to DBA/1 mice during the early induction phase of collagen-induced arthritis and demonstrated therapeutic efficacy of alpha-GalCer when administered early rather than late during the disease. Surprisingly, the Th1-polarizing analog alpha-C-GalCer also conferred protection. Furthermore, a biphasic role of IFN-gamma in the effect of iNKT cell stimulation was observed. Whereas in vivo neutralization of IFN-gamma release induced by either alpha-GalCer or alpha-C-GalCer early during the course of disease resulted in partial improvement of clinical arthritis symptoms, blockade of IFN-gamma release later on resulted in a more rapid onset of arthritis. Although no phenotypic changes in conventional T cells, macrophages, or APCs could be detected, important functional differences in T cell cytokine production in serum were observed upon polyclonal T cell activation, 2 wk after onset of arthritis. Whereas alpha-GalCer-treated mice produced significantly higher amounts of IL-10 upon systemic anti-CD3 stimulation compared with PBS controls, T cells from alpha-C-GalCer-treated mice, by contrast, produced substantially lower levels of cytokines, suggesting the involvement of different protective mechanisms. In conclusion, these findings suggest long-term, ligand-specific, time-dependent, and partially IFN-gamma-dependent immunomodulatory effects of iNKT cells in collagen-induced arthritis.     

Production of IL-12 and IL-18 in human dendritic cells upon infection by Listeria monocytogenes

Dendritic cells (DCs) are major antigen-presenting cells of the immune system, which need to be activated in order to initiate an immune response. Here, we describe the immunostimulatory effects on human monocyte-derived DCs observed upon infection with Listeria monocytogenes or after treatment with listerial lipoteichoic acid (LTA) and lipopolysaccharide (LPS), respectively. All stimuli caused upregulation of costimulatory molecules, induced T-cell proliferative responses and secretion of cytokines in vitro. Infection of DCs with L. monocytogenes induced release of interleukin (IL)-12 and IL-18. In contrast treatment with purified listerial LTA yielded high levels of IL-18 release, but only minimal IL-12 production. Treatment of DCs with LPS conversely induced significant amounts of IL-12 production, but no IL-18. The release of both stimulating cytokines IL-12 and IL-18 upon infection with entire bacteria suggests that attenuated strains of L. monocytogenes may be a valuable tool for subunit vaccine delivery.     

IFN-gamma enhances bovine macrophage responsiveness to Mycobacterium bovis: Impact on bacterial replication, cytokine release and macrophage apoptosis

We sought to determine the impact of bovine IFN-gamma on the interaction between Mycobacterium bovis and bovine macrophages. Bovine macrophages released small amounts of nitric oxide (NO), TNF-alpha, IL-1beta and IL-12 upon infection with bacille Calmette-Guérin (BCG). Prior pulsing of cells with IFN-gamma significantly enhanced the release of NO and IL-12. Infection of bovine macrophages with virulent M. bovis led to the release of higher levels of pro-inflammatory mediators, compared to levels released upon BCG infection. IFN-gamma treatment of macrophages enhanced the release of pro-inflammatory mediators, but did not modify bacterial replication in M. bovis-infected macrophages. Treatment of macrophages with a combination of IFN-gamma and LPS led to a reduction in bacterial replication. Infected cells treated with IFN-gamma/LPS progressed mostly through an apoptotic pathway, whereas untreated infected cells eventually died by necrosis. Agents that prevented the acquisition of bacteriostatic activity by activated macrophages also prevented the induction of apoptosis in infected macrophages (IL-10 and neutralizing anti-TNF-alpha). We conclude that virulent M. bovis is a major determinant of release of pro-inflammatory cytokines by macrophages. IFN-gamma amplifies the macrophage cytokine release in response to M. bovis. Induction of apoptosis is closely linked to the emergence of macrophage resistance to M. bovis replication, which is dependent on endogenous TNF-alpha release.     

Interactions of mononuclear cells of peripheral blood with islet cells in vitro

Peripheral blood mononuclear cells from Type I diabetic patients health donors or Wistar rats were cultured with rat islet cells for 18 h. Then TNF content of the medium and basal and stimulated insulin release were determined. Mononuclear cells from both healthy donors and diabetic patients could inhibit the insulin release with no correlation to TNF content. Addition of lipopolysaccharide resulted in a 5-7 times increase of TNF content of the medium followed by a more pronounced inhibition of insulin release. Rat mononuclear cells inhibited the beta-cell function almost completely and initially produced large amounts of TNF. The data indicate that inhibition of insulin release by blood mononuclear cells in vitro does not reflect anti beta-cell specific cellular immunity, involves cytokines and, probably depends upon the initial properties of the cells.     

Second messenger function of nicotinic acid adenine dinucleotide phosphate revealed by an improved enzymatic cycling assay

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent activator of Ca2+ release from intracellular stores known today. Although recent reports have suggested an important function of NAADP in human T lymphocytes, direct evidence for receptor-induced formation of NAADP is yet missing in these cells. Thus, we developed a highly sensitive and specific enzyme assay capable of quantifying low fmol amounts of NAADP. In unstimulated T cells, the NAADP concentration amounted to 4.4 +/- 1.6 nm (0.055 +/- 0.028 pmol/mg of protein). Stimulation of the cells via the T cell receptor/CD3 complex resulted in biphasic elevation kinetics of cellular NAADP levels and was characterized by a bell-shaped concentration-response curve for NAADP. In contrast, the NAADP concentration was elevated neither upon activation of the ADP-ribose/TRPM2 channel Ca2+ signaling system nor by an increase of the intracellular Ca2+ concentration upon thapsigargin stimulation. T cell receptor/CD3 complex-mediated NAADP formation was dependent on the activity of tyrosine kinases because genistein completely blocked NAADP elevation. Thus, we propose a regulated formation of NAADP upon specific stimulation of the T cell receptor/CD3 complex, suggesting a function of NAADP as a Ca2+-mobilizing second messenger during T cell activation.     

Role of TGF-beta 1 on the IgE-dependent anaphylaxis reaction

TGF-beta1 is a member of a family of polypeptide factors that control proliferation, differentiation, chemotaxis, and other functions in many cell types. TGF-beta1 has been shown to inhibit many immunologic functions. However, here we report that TGF-beta1 has an important role in the elicitation of IgE-dependent allergic reactions. The synthetic antisense TGF-beta1 oligonucleotides dose-dependently inhibit passive cutaneous anaphylaxis (PCA) reaction and histamine release from the mast cells activated by anti-DNP IgE in rats. The level of cAMP in mast cells, when antisense TGF-beta1 oligonucleotides was added, significantly increased approximately 7-fold compared with that of basal cells. The antisense TGF-beta1 oligonucleotides also had a significant inhibitory effect on anti-DNP IgE-induced TNF-alpha release from mast cells. In situ hybridization analysis showed that the PCA reaction sites treated with antisense TGF-beta1 oligonucleotides exhibited no detectable levels of TGF-beta1 and L-histidine decarboxylase mRNA after anti-DNP IgE stimulation, whereas the PCA reaction sites treated with sense TGF-beta1 oligonucleotides possessed significant amounts of their mRNA. Additionally, neutralizing Ab to TGF-beta1 blocked the PCA reaction significantly, but its Ab did not inhibit peritoneal mast cell-released histamine upon treatment with anti-DNP IgE. Our results suggest that TGF-beta1 is critical to the development of IgE-dependent anaphylaxis reactions.     

An artificial antigen-presenting cell with paracrine delivery of IL-2 impacts the magnitude and direction of the T cell response

Artificial antigen-presenting cells (aAPCs) are an emerging technology to induce therapeutic cellular immunity without the need for autologous antigen-presenting cells (APCs). To fully replace natural APCs, an optimized aAPC must present antigen (signal 1), provide costimulation (signal 2), and release cytokine (signal 3). Here we demonstrate that the spatial and temporal characteristics of paracrine release of IL-2 from biodegradable polymer aAPCs (now termed paAPCs) can significantly alter the balance in the activation and proliferation of CD8+ and CD4+ T cells. Paracrine delivery of IL-2 upon T cell contact with paAPCs induces significant IL-2 accumulation in the synaptic contact region. This accumulation increases CD25 (the inducible IL-2 Rα chain) on responding T cells and increases proliferation of CD8+ T cells in vitro to levels 10 times that observed with equivalent amounts of bulk IL-2. These CD8+ T cell responses critically depend upon close contact of T cells and the paAPCs and require sustained release of low levels of IL-2. The same conditions promote activation-induced cell death in CD4+ T cells. These findings provide insight into the response of T cell subsets to paracrine IL-2.     

Enhancement of cell-cell contact by a nonmitogenic lectin increases blastogenic response and IL-2 release by mitogen-stimulated mouse thymocytes

We have examined the influence of peanut agglutinin (PNA), a lectin which agglutinates but does not stimulate mouse thymocytes, on the responsiveness of these cells to concanavalin A (Con A) or galactose oxidase stimulation. Binding low amounts of PNA on unseparated mouse thymocytes pretreated with neuraminidase highly enhances the mitogenic response and the level of interleukin 2 release in the culture medium upon Con A stimulation. We have shown that PNA present on the cell surface acts as a crosslinking agent which favors intercellular binding between accessory cells (macrophages) and thymocytes, leading through this enhanced cooperation by cell-cell contact to an enhanced blastogenic response.     

Biochemical analysis of desensitization of mouse mast cells

Biochemical mechanisms of desensitization were explored by using peritoneal mouse mast cells saturated with monoclonal mouse IgE anti-DNP antibody. It was found that a 1-min incubation of the sensitized cells with 0.01 micrograms/ml DNP-HSA in the absence of Ca2+ was sufficient to desensitize the cells completely. The treated cells failed to release a detectable amount of histamine upon incubation with an optimal concentration (0.1 to 1.0 micrograms/ml) of DNP-HSA and Ca2+. Determination of the number of antigen molecules bound to mast cells revealed that only a small (less than 10%) fraction of cell-bound IgE antibody molecules reacted with desensitizing antigen, and that desensitized cells and untreated (sensitized) cells could bind comparable amounts of antigen upon incubation with rechallenging antigen. However, the binding of antigen molecules to desensitized cells failed to induce any of the early biochemical events, i.e., phospholipid methylation, cAMP rise, and 45Ca uptake, as well as histamine release. It was also found that intracellular cAMP levels in desensitized cells were comparable to those in sensitized cells. Desensitization by a suboptimal concentration of DNP-HSA was prevented by inhibitors of methyltransferases, such as 3-deaza adenosine plus L-homocysteine thiolactone. Sensitized cells pretreated with 0.01 micrograms/ml DNP-HSA in the absence of Ca2+ and in the presence of the methyltransferase inhibitors responded to an optimal concentration of antigen for histamine release when they were rechallenged in the presence of Ca2+. Inhibition of desensitization by methyltransferase inhibitors was reversed by the addition of S-adenosyl-L-methionine to the system. The results indicated that the activation of methyltransferases, induced by receptor bridging, is involved in the process of desensitization. Desensitization was inhibited by reversible inhibitors of serine proteases, such as p-aminobenzamidine, indole, and synthesized substrates of rat mast cell proteases. It was also found that diisopropylfluorophosphate (DFP), an irreversible inhibitor of serine proteases, completely blocked desensitization at the concentration of 10 to 40 nM. This concentration of DFP did not affect the antigen-induced histamine release, whereas 100- to 1000-fold higher concentrations of DFP did inhibit histamine release. The results suggest that serine proteases are involved in both the induction of histamine release and desensitization, and that the protease involved in desensitization is distinct from that involved in triggering histamine release.     

Subcellular location of a soluble factor that stimulates DNA replication in permeable animal cells

The enzymatic isolation of cells with bacterial collagenase has proved to be a powerful technique for the study of a wide variety of tissues. Unfortunately, for some applications such as the isolation of cells from membranous bone, the cellular damage that results from the exposure of the cells to cytotoxic contaminants of bacterial collagenase has limited the usefulness of this approach. The use of chromatographically purified collagenase alone is often ineffective or very slow to release cells from tissue. We have found that two enzymes are necessary and sufficient to isolate cells from neonatal mouse calvaria: purified collagenase and neutral protease. These two enzymes can be chromatographically purified on a preparative scale to yield 100 mg amounts of each enzyme. The purified enzymes can be recombined in amounts which will digest calvaria at the same rate as the crude bacterial collagenase from which they were derived. The cells that are isolated using the purified enzymes are undamaged, as indicated by the measurement of their equilibrium density on gradients of Ficoll and sodium metrizoate. Cells isolated with crude collagenase never reach an equilibrium density upon isopyknic centrifugation, whereas cells isolated with the purified enzymes reach an equilibrium density of 1.074 g/ml in 90 min.     

Isolation from subcellular preparation of a mediator of hypoglycaemic hGH peptides

This study demonstrated the release into the incubation medium of a cellular mediator from isolated fat adipocytes and hepatocytes after treatment with the hypoglycaemic fragment of human growth hormone, hGH 6-13. The activity of the putative mediator observed in the cell "ghosts" of both liver and rat cells suggests that the active component is likely to be derived from plasma membranes and has an ubiquitous cellular distribution. The hGH fragment-induced release of the mediator from plasma membranes depends upon the physiological status of the animals. Liver plasma membranes of starved rats yield significantly higher levels of the cellular mediator in response to treatment with hGH 6-13. The studies of the physiological factors influencing the release of the material from cellular systems clearly enhance the production of adequate amounts of the cellular mediator for molecular characterization. The precise chemical nature and the physiological role of the hGH cellular mediator are currently unknown.     

Lactate interferes with ATP release from red blood cells

Upon exposure to low Po(2), the red blood cells of most species, including humans, release increased amounts of ATP that ultimately serves as a regulator of vascular tone matching oxygen supply with demand. In pathological conditions such as malaria and sepsis, a maldistribution of perfusion exists with its severity often correlated with the extent of elevation of serum lactate frequently in the absence of an alteration in pH. We hypothesized that the increased levels of lactate might impair the ability of red blood cells to appropriately respond to conditions of low Po(2), thus preventing its important blood flow regulatory role. Using an in vitro system and rabbit red blood cells, we evaluated the capacity of cells incubated with lactate to release increased amounts of ATP in response to acute exposure to low Po(2). We found that in the presence of lactate, the red blood cells did not release ATP. However, when sodium dichloroacetate, a drug used clinically to lower blood lactate levels, was added, ATP release was restored to levels that were not different from that of control cells (no lactate), even though intracellular levels of ATP were not. These results support the presence of a distinct flow regulatory pool of ATP within the red blood cell that can be independently regulated, and that lactate interferes with the ATP production within this pool, thereby diminishing the amount of ATP available for release on exposure to low Po(2). Therefore, if lactate levels can be reduced, the vascular regulatory capacity of the red blood cell should be restored, thus enabling the appropriate matching of oxygen supply with oxygen demand.     

Influence of divalent cations upon complement-mediated enzyme release from human polymorphonuclear leukocytes.

The complement component, C5a provokes the selective release of granule-associated enzymes from the intact, viable cytochalasin B-treated human polymorphonuclear leukocytes (PMN) in the absence of phagocytosis or cellular adherence to surfaces. Consquently, in this experimental system the influence of divalent cations on these two processes can be disregarded and their effects on enzymes secretion can be studied directly. Cytochalasin B-treated PMN exposed to C5a in calcium and magnesium-free media consistently secreted significant amounts of the granule-associated enzymes, beta-glucuronidase and lysozyme. The basal secretory response was not diminished if cells were preincubated with 5.0 mM EDTA, nor was it influenced if 1.0 mm or 2.0 mM EDTA were present in the reaction mixtures. The addition of calcium (up to 1.5 to 2.0 mM) produced a concentration-dependent enhancement of beta-glucuronidase release, whereas increasing amounts of calcium (above 2.0 mM) inhibited secretion of this enzyme. Lysozyme release was similarly enhanced by the addition of calcium, but inhibition with high concentrations was not observed. Calcium per se, in the absence of C5a, provoked only the release of lysozyme from these cells. The effects of calcium upon enzyme release were not associated with alterations in the state of assembly of cytoplasmic microtubules. These findings provide another example of the role of calcium in "stimulus-secretion coupling" and provide evidence that exocytosis of various granules in human PMN is regulated by independent mechanisms involving calcium.     

Sphingomyelin exhibits greatly enhanced protection compared with egg yolk phosphatidylcholine against detergent bile salts

Inclusion of phosphatidylcholine within bile salt micelles protects against bile salt-induced cytotoxicity. In addition to phosphatidylcholine, bile may contain significant amounts of sphingomyelin, particularly under cholestatic conditions. We compared protective effects of egg yolk phosphatidylcholine (similar to phosphatidylcholine in bile), egg yolk sphingomyelin (mainly 16:0 acyl chains) and dipalmitoyl phosphatidylcholine against taurocholate in complementary in vitro studies. Upon addition of taurocholate-containing micelles to sonicated egg yolk phosphatidylcholine vesicles, subsequent micellization of the vesicular bilayer proved to be retarded when phospholipids had also been included in these micelles in the rank order: egg yolk phosphatidylcholine < dipalmitoyl phosphatidylcholine < sphingomyelin. Hemolysis of erythrocytes and LDH release by CaCo-2 cells after addition of taurocholate micelles were strongly reduced by including small amounts of sphingomyelin or dipalmitoyl phosphatidylcholine in these micelles (PL/(PL + BS) >/= 0.1), whereas egg yolk phosphatidylcholine provided less protection. Amounts of non-phospholipid-associated bile salts (thought to be responsible for cytotoxicity) in egg yolk phosphatidylcholine-containing micelles were significantly higher than in corresponding sphingomyelin- or dipalmitoyl phosphatidylcholine-containing micelles (tested at PL/(PL + BS) ratios 0.1, 0.15, and 0.2). LDH release upon incubation of CaCo-2 cells with taurocholate simple micelles at these so-called "intermixed micellar-vesicular" concentrations was identical to LDH release upon incubation with corresponding taurocholate-phospholipid mixed micelles. In conclusion, we found greatly enhanced protective effects of sphingomyelin and dipalmitoyl phosphatidylcholine compared to egg yolk phosphatidylcholine against bile salt-induced cytotoxicity, related to different amounts of non-phospholipid-associated bile salts. These findings may be relevant for protection against bile salt-induced cytotoxicity in vivo.     

Leishmania (Viannia) braziliensis: human mast cell line activation induced by logarithmic and stationary promastigote derived-lysates

Herein we investigate the ability of live promastigotes and total lysate of Leishmania (Viannia) braziliensis, derived from parasites in the logarithmic (L-Lb) or stationary phase (S-Lb), to induce human mast cell line (HMC-1) activation. In comparison with medium-treated cells, a significant histamine release was observed in HMC-1 cultures stimulated with S-Lb. Lipophosphoglycan also induced histamine release by HMC-1 cells. In immunocytochemical assays, we found a marked staining for tryptase in medium-treated HMC-1 cells, however, stimulation with L-Lb or S-Lb caused a marked decrease in the color reaction as well as in the number of tryptase-positive cells. L-Lb and S-Lb induced an evident decrease in the intracellular expression of IL-4 but not IL-12. Live stationary promastigotes were able to induce high levels of IL-4 release in HMC-1 cultures. Furthermore, these cells released significant amounts of IL-12 when incubated with both types of live promastigotes. These results indicate that L. (V.) braziliensis promastigotes differ in their ability to induce direct human mast cells activation, according to the growth phase of the parasite. Furthermore, the release of pro-inflammatory mediators and cytokines could represent an important phenomenon that might favor the initial establishment of the infection.     

Testosterone inhibits expression of inducible nitric oxide synthase in murine macrophages

We investigated the effect of testosterone, the main sexual steroid hormone in men, upon inducible nitric oxide synthesis in murine macrophages. Incubation of murine macrophages (RAW 264.7 cells) stimulated by bacterial lipopolysaccharide (2 microg/ml) with increasing amounts of testosterone (0.1-40 microM) showed a dose dependent inhibition of inducible nitric oxide synthesis. Inducible nitric oxide synthase protein expression was reduced in a dose dependent manner as revealed by immunoblotting when cells were incubated with increasing amounts of testosterone. This was associated with a decline in iNOS mRNA-levels as determined by competitive semiquantitative PCR. As nitric oxide plays an important role in immune defense and atherosclerosis prevention, testosterone-induced iNOS inhibition could lead to an elevated risk of infection as well as to the development of atherosclerotic lesions.     

Anion homeostasis is important for non-lytic release of BK polyomavirus from infected cells

BK polyomavirus (BKPyV) is a member of a family of potentially oncogenic viruses, whose reactivation can cause severe pathological conditions in transplant patients, leading to graft rejection. As with many non-enveloped viruses, it is assumed that virus release occurs through lysis of the host cell. We now show the first evidence for a non-lytic release pathway for BKPyV and that this pathway can be blocked by the anion channel inhibitor DIDS. Our data show a dose-dependent effect of DIDS on the release of BKPyV virions. We also observed an accumulation of viral capsids in large LAMP-1-positive acidic organelles within the cytoplasm of cells upon DIDS treatment, suggesting potential late endosome or lysosome-related compartments are involved in non-lytic BKPyV release. These data highlight a novel mechanism by which polyomaviruses can be released from infected cells in an active and non-lytic manner, and that anion homeostasis regulation is important in this pathway.     

Localization of alkaline phosphatase in three gram-negative rumen bacteria

Of the three species (Bacteroides ruminicola, B. succinogenes, and Megasphaera elsdenii) of anaerobic gram-negative rumen bacteria studied, only B. ruminicola produced significant amounts of alkaline phosphatase. This enzyme, which is constitutive, showed a greater affinity for p-nitrophenylphosphate than for sodium-beta-glycerophosphate and was shown to be located exclusively in the periplasmic space of log-phase cells. Small amounts of this enzyme were released from these cells in stationary-phase cultures, but washing in 0.01 M MgCl(2) and the production of spheroplasts by using lysozyme in 0.01 M MgCl(2) did not release significant amounts of the enzyme. Exposure to 0.2 M MgCl(2) did not release significant amounts of the periplasmic alkaline phosphatase of the cell, and when these cells were spheroplasted with lysozyme in 0.2 M MgCl(2) only 25% of the enzyme was released. Spheroplasts were formed spontaneously in aging cultures of B. ruminicola, but even these cells retained most of their periplasmic alkaline phosphatase. It was concluded that the alkaline phosphatase of B. ruminicola is firmly bound to a structural component within the periplasmic area of the cell wall and that the enzyme is released in large amounts only when the cells break down. The behavior of alkaline phosphatase in this bacterium contrasts with that of conventional periplasmic enzymes of aerobic bacteria, which are released upon conversion into spheroplasts by lysozyme and ethylenediaminetetraacetic acid and by other types of cell wall damage. All three species of bacteria studied here, as well as bacteria found in mixed populations in the rumen, have thick, complex layers external to the double-track layer of their cell walls. In addition, B. ruminicola produces a loose extracellular material.     

Bronchoalveolar cell activation after inhalation of a bronchoconstricting agent

Airway inflammation is thought to be an important determinant of bronchoconstriction and bronchial hyperreactivity. We have recently demonstrated that bronchoconstriction induced by an aqueous extract of cotton bracts (CBE) is associated with bronchoalveolar complement activation, release of polymorphonuclear neutrophil (PMN) chemoattractants by pulmonary cells, and increased numbers of bronchoalveolar lavage PMN's. In the present study we performed bronchoalveolar lavage (BAL) on subjects after CBE or control (saline) challenge and examined whether BAL cells were activated in vitro to produce other inflammatory agonists. After CBE administration, cultured BAL cells released increased amounts of the reactive O2 species, superoxide (O2-.), and the cyclooxygenase products prostaglandin E2 and thromboxane B2. Although none of these in vitro parameters of BAL cell activation appeared to correlate with the degree of bronchoconstriction induced by CBE, BAL fluid levels of thromboxane B2 were also increased after CBE administration and in vivo amounts of this eicasanoid did correlate with the degree of bronchoconstriction induced by CBE (r = 0.50, P less than 0.04). Finally, although cell culture supernatants were highly chemotactic for PMN's, concentrations of leukotriene B4 were not increased, suggesting other chemotaxins were released by BAL cells in this setting. We conclude that CBE administration activates bronchoalveolar cells to release reactive O2 species and cyclooxygenase products that may be important in the bronchoconstricting response to CBE.