ADP and other metabolites released from Acanthamoeba castellanii lead to human monocytic cell death through apoptosis and stimulate the secretion of proinflammatory cytokines

Monocytes/macrophages are thought to be involved in Acanthamoeba infections. The aim of this work was to study whether soluble metabolites (ADP and other compounds) released by Acanthamoeba castellanii trophozoites could induce morphological and biochemical changes in human monocytic cells in vitro. We demonstrate here that ADP constitutively released in the medium by A. castellanii, interacting with specific P2y(2) purinoceptors expressed on the monocytic cell membrane, caused a biphasic rise in [Ca(2+)](i), morphological changes characteristics of cells undergoing apoptosis, caspase-3 activation, and secretion of tumor necrosis factor alpha (TNF-alpha). The same results were found in monocytes exposed to purified ADP. Cell damage and TNF-alpha release induced by amoebic ADP were blocked by the P2y(2) inhibitor suramin. Other metabolites contained in amoebic cell-free supernatants, with molecular masses of, respectively, >30 kDa and between 30 and 10 kDa, also caused morphological modifications and activation of intracellular caspase-3, characteristics of programmed cell death. Nevertheless, mechanisms by which these molecules trigger cell damage appeared to differ from that of ADP. In addition, other amoebic thermolable metabolites with molecular masses of <10 kDa caused the secretion of interleukin-1beta. These findings suggest that pathogenic free-living A. castellanii by release of ADP and other metabolites lead to human monocytic cell death through apoptosis and stimulate the secretion of proinflammatory cytokines.     

Purinergic activation of BK channels in clonal kidney cells (Vero cells)

We have studied the activation of a high-conductance channel in clonal kidney cells from African green monkey (Vero cells) using patch-clamp recordings and microfluorometric (fura-2) measurements of cytosolic Ca2+. The single-channel conductance in excised patches is 170 pS in symmetrical 140 mM KCl. The channel is highly selective for K+ and activated by membrane depolarization and application of Ca2+ to the cytoplasmatic side of the patch. The channel is, thus, a large-conductance Ca2+-activated K+ channel (BK channel). Cell-attached recordings revealed that the channel is inactive in unstimulated cells. Extracellular application of less than 0.1 microM ATP transiently increased the cytosolic Ca2+ concentration ([Ca2+]i) to about 550 nM, and induced membrane hyperpolarization caused by Ca2+-activated K+ currents. ATP stimulation also activated BK channels in cell-attached patches at both the normal-resting potential and during membrane hyperpolarization. The increase in [Ca2+]i was owing to Ca2+ release from internal stores, suggesting that Vero cells express G-protein-coupled purinergic receptors (P2Y) mediating IP3-induced release of Ca2+. The P2Y receptors were sensitive to both uracil triphosphate (UTP) and adenosine diphosphate (ADP), and the rank of agonist potency was ATP > UTP >/= ADP. This result indicates the presence of both P2Y1 and P2Y2 receptors or a receptor subtype with untypical agonist sensitivity. It has previously been shown that hypotonic challenge activates BK channels in both normal and clonal kidney cells. The subsequent loss of KCl may be an important factor in cellular volume regulation. Our results support the idea of an autocrine role of ATP in this process. A minute release of ATP induced by hypotonically evoked membrane stretch may activate the P2Y receptors, subsequently increasing [Ca2+]i and thus causing K+ efflux through BK channels.     

Adenovirus ADP protein (E3-11.6K), which is required for efficient cell lysis and virus release,interacts with human MAD2B

The human subgroup C adenovirus (Ad) protein named adenovirus death protein (ADP) (previously named E3-11.6K) is synthesized at very late stages of infection when it mediates efficient lysis of cells and release of adenovirus to infect other cells. ADP is an integral membrane N-linked, O-linked palmitoylated glycoprotein of 101 amino acids (aa) that localizes to the nuclear membrane, endoplasmic reticulum (ER), and Golgi. It has a single membrane spanning region (roughly aa 40-60) and is oriented with aa 1-40 in the lumen and aa 61-101 in the nucleoplasm and cytoplasm. Using aa 61-101 of Ad2 ADP as bait in a yeast two-hybrid screen, we isolated a cDNA for a 211-aa protein that initially was not in the database but has now been published by others with the names human MAD2B, MAD2L2, and REV7. ADP binds strongly to human MAD2B not only in yeast but also in GST pull-down experiments and in coimmunoprecipitations of ADP and MAD2B synthesized in vitro or in vivo. ADP mutants with deletions throughout the bait region do not interact with human MAD2B, whereas a Pro69Pro70 to Ala69Ala70 mutant in the "basic-proline" domain of ADP does interact. Northern blot analyses indicate that human MAD2B is expressed ubiquitously. Human MAD2B is about 25% identical to human MAD2, a spindle assembly checkpoint protein. Two human A549 cell lines were made that constitutively overexpress MAD2B. Wild-type adenovirus lyses these cells significantly more slowly than it lyses parental A549 cells, raising the possibility that ADP and MAD2B act in opposition and suggesting that the ADP-MAD2B interaction is biologically relevant.     

Development of a radioimmunoassay for human milk-fat-globule membrane glycoprotein. Its quantitation in spent media from both primary and established mammary and non-mammary epithelial cell lines

A radioimmunoassay (RIA) was developed for the quantitative measurement of a glycoprotein which was purified from human milk-fat-globule membrane (MFGM) and termed MFGM-pg 70. The assay with a sensitivity of detecting 30 ng of MFGM-pg 70/ml was employed to quantitate the levels of MFGM-gp 70 shed in supernatants from primary cultures of normal and malignant human breast cells and from various established cell lines of human mammary including myoepithelial and fibroblast and non-mammary malignant epithelial cells. The RIA for MFGM-gp 70 showed that the amount of antigen shed was much higher in supernatants from normal mammary epithelial cells compared with their malignant counterparts grown in primary culture and with those from established cell lines of malignant mammary epithelial cells. No detectable antigen was found in supernatants from cultures of normal myoepithelial-like cell lines or primary cultures of fibroblast cells from breast, or cell lines of squamous carcinomas of head and neck and tongue, renal cell carcinoma and teratoma. Trypsinization of mammary epithelial cells from both primary and established lines resulted in the release of most of the antigen from the surface of the cells, suggesting the presence of this molecule on the cell's surface. Following trypsinization, approximately 99% of the cells was viable, indicating that the release of the antigen in supernatants was due to shedding and not cell death. The levels of MFGM-pg 70 in spent media were unaffected by the lactogenic hormones such as prolactin or insulin. The RIA for MFGM-gp 70 provides a sensitive and quantitative means to in vitro study the synthesis of a membrane glycoprotein from human mammary epithelium.     

Receptor-mediated facilitation of cell volume regulation by swelling-induced ATP release in human epithelial cells

Osmotic swelling induces the release of intracellular ATP in a number of cell types. In the immediate vicinity of the cell surface, released ATP has been shown to reach a concentration high enough to stimulate P2-purinergic receptors in a human epithelial cell line, Intestine 407. The role of released ATP in the regulatory volume decrease (RVD) after cell swelling was thus studied in Intestine 407 cells. The RVD was suppressed by an ATP hydrolyzing enzyme, apyrase, or by a purinergic receptor antagonist, suramin. Extracellular application of ATP accelerated the RVD rate in a concentration-dependent manner. An increase in the cytosolic free-Ca(2+) concentration was induced by a hypotonic challenge, and the swelling-induced Ca(2+) response was partially suppressed by apyrase or suramin. A rise in cytosolic Ca(2+) was also induced by extracellular application of ATP or UTP, but not ADP, 2-methylthio-ATP or alpha,beta-methylene ATP. The ATP-induced Ca(2+) response was blocked by suramin. Therefore, it is concluded that RVD is facilitated by ATP, which is released upon cell swelling, by augmenting intracellular Ca(2+) rise via the stimulation of purinergic (P2Y(2)) receptors in the human epithelial cell.     

Müller cell Ca2+ waves evoked by purinergic receptor agonists in slices of rat retina

We have measured agonist evoked Ca2+ waves in Müller cells in situ within freshly isolated retinal slices. Using an eye cup dye loading procedure we were able to preferentially fill Müller glial cells in retinal slices with calcium green. Fluorescence microscopy revealed that bath perfusion of slices with purinergic agonists elicits Ca2+ waves in Müller cells, which propagate along their processes. These Ca2+ signals were insensitive to tetrodotoxin (TTX, 1.0 microM) pretreatment. Cells were readily identified as Müller cells by their unique morphology and by subsequent immunocytochemical labeling with glial fibrillary acidic protein antibodies. While cells never exhibited spontaneous Ca2+ oscillations, purinoreceptor agonists, ATP, 2 MeSATP, ADP, 2 MeSADP, and adenosine readily elicited Ca2+ waves. These waves persisted in the absence of [Ca2+]o but were abolished by thapsigargin pretreatment, suggesting that the purinergic agonists tested act by releasing Ca2+ from intracellular Ca2+ stores. The rank order of potency of different purines and pyrimidines for inducing Ca2+ signals was 2 MeSATP = 2MeSADP > ADP > ATP > alphabetameATP = uridine triphosphate (UTP) > uridine diphosphate (UDP). The Ca2+ signals evoked by ATP, ADP, and 2 MeSATP were inhibited by reactive blue (100 microM) and suramin (200 microM), and the adenosine induced signals were abolished only by 3,7-dimethyl-1-propargylxanthine (200 microM) and not by 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine) or 8-cyclopentyl-1,3-dipropylxanthine at the same concentration. Based on these pharmacological characteristics and the dose-response relationships for ATP, 2 MeSATP, 2 MeSADP, ADP, and adenosine, we concluded that Müller cells express the P1A2 and P2Y1 subtypes of purinoceptors. Analysis of Ca2+ responses showed that, similar to glial cells in culture, wave propagation occurred by regenerative amplification at specialized Ca2+ release sites (wave amplification sites), where the rate of Ca2+ release was significantly enhanced. These data suggest that Müller cells in the retina may participate in signaling, and this may serve as an extra-neuronal signaling pathway.     

Polarized expression of Tamm-Horsfall protein by renal tubular epithelial cells activates human granulocytes

In renal bacterial infections granulocytes are of major importance in the primary immune defense against invading pathogens. However, the mechanisms of granulocytic activation in renal interstitial invasion have not been clarified. Renal tubular epithelial cell mechanisms inducing granulocytic activation and bacterial killing may include tubular cell expression of Tamm-Horsfall protein (THP), a urinary protein that is known to enhance cytokine expression in monocytes. We studied the role of THP in granulocytic activation. A strong binding of THP to human granulocytes was demonstrated by fluorescence-activated cell sorter analysis. Urinary THP and supernatants of THP-expressing cultured tubular epithelial cells (MDCK) enhanced interleukin-8 (IL-8) expression by human granulocytes. Renal tubular cells growing polarized on polycarbonate membranes were used to study apical versus basal THP expression. By electron microscopy THP immunoreactivity was exclusively found on the apical surfaces of tubular cells and was absent on the basolateral cell membrane. In the apical cell culture compartment we found significantly more stimulatory activity for granulocytic IL-8 expression. CD62L, a selectin less expressed in activated granulocytes, was decreased in granulocytes incubated with urinary THP and in supernatants of THP-producing renal tubular cells but not in supernatants from THP-negative cells. Again, the effect on CD62L expression was found only in apical culture media and was absent in the basal compartment. In summary our data give evidence that renal tubular cell THP expression may be relevant in kidney diseases since THP is a potent activator of human granulocytes. The regulation of apical versus basal THP expression and release in vivo may be crucial in the induction of the inflammatory response, e.g., in bacterial renal diseases.     

Necessity for interaction between adherent and non-adherent rat spleen cells for the generation of a suppressor factor of NK activation

Whole rat spleen cell populations in culture release constitutively a factor which suppresses the NK activation in response to interleukin-2. Culture supernatants derived from adherent (AD) and non-adherent (NAD) cell preparations obtained from rat spleen cells, did not have a suppressor activity comparable to that present in the culture supernatants of whole spleen cells. When reconstituted mixtures of the AD and NAD cells were cultured, high levels of suppressor activity could again be demonstrated in the culture supernatants, indicating that some kind of co-operation between AD and NAD cells was needed for efficient generation of the suppressor. Non-suppressive culture supernatants derived from NAD cells became suppressive when AD cells were cultured in them. On the other hand, relatively little suppressor activity was generated when NAD cells were cultured in non-suppressive culture supernatants from AD cells. Our results seem to indicate that NAD cells may release some agent which is not itself a suppressor but either (a) induces the generation of suppressor activity from AD cells, or (b) is "processed" into a suppressor agent by AD cells.     

Growth inhibitory effects of ATP and its derivatives on human fibroblasts immortalized with 60Co-gamma rays

In our previous study (Katayama B et al, Int J Mol Med 2: 603-606, 1998), cell growth inhibition caused by ATP added to cultures was found to be greater in immortalized human fibroblasts than in the normal human fibroblasts. Since it has been reported that ATP affects cells via P2-purinergic receptors, growth inhibitory effects of ATP and its derivatives on immortalized human fibroblasts were investigated in the present study in order to learn what type of receptors are involved in ATP cytotoxicity. The ATP derivatives used in this study were: ATP, ADP, beta, gamma-methyleneadenosine 5'-triphosphate (MeATP), 2' & 3'-o-(4-benzoylbenzoyl) adenosine, triethylammonium salt (BzATP), adenosine 5'-o-(3-thiotriphosphate) (ATPgammaS), 2-methylthioadenosine 5'-triphosphate (2-MeSATP) and UTP. The extent of cytotoxicity induced by these drugs was found to be in the order of: ATP=ADP>ATPgammaS>MeATP=BzATP. On the other hand, neither 2-MeSATP nor UTP showed any cytotoxicity. These findings indicate that ATP may exert the cell growth inhibition by certain kinds of signal transduction via P2x or P2y purinergic receptors which affect intrinsic channels/pores of cell membrane and/or G protein activation. As a result, intracellular elevation in the concentrations of ions such as calcium and potassium, membrane depolarization, loss of endogenous ions/metabolites, and activation of inositol phospholipid-specific phospholipase C may occur. Actually, a dihydropyridine calcium channel blocker, nifedipine, and an ATP-sensitive K+-channel blocker, glybenclamide, reduced the growth inhibitory effects of ATP on the cells to some extent. The growth inhibition caused by ATP was not due to apoptosis or induction of a cyclin/CDK kinase inhibitor, P21.     

Shiga toxin 2 and lipopolysaccharide induce human microvascular endothelial cells to release chemokines and factors that stimulate platelet function

Shiga toxins (Stxs) produced by Shigella dysenteriae type 1 and enterohemorrhagic Escherichia coli are the most common cause of hemolytic-uremic syndrome (HUS). It is well established that vascular endothelial cells, mainly those located in the renal microvasculature, are targets for Stxs. The aim of the present research was to evaluate whether E. coli-derived Shiga toxin 2 (Stx2) incubated with human microvascular endothelial cells (HMEC-1) induces release of chemokines and other factors that might stimulate platelet function. HMEC-1 were exposed for 24 h in vitro to Stx2, lipopolysaccharide (LPS), or the Stx2-LPS combination, and chemokine production was assessed by immunoassay. More interleukin-8 was released than stromal cell-derived factor 1alpha (SDF-1alpha) or SDF-1beta and RANTES. The Stx2-LPS combination potentiated chemokine release, but Stx2 alone caused more release of SDF-1alpha at 24 h than LPS or Stx2-LPS did. In the presence of low ADP levels, HMEC-1 supernatants activated platelet function assessed by classical aggregometry, single-particle counting, granule secretion, P-selectin exposure, and the formation of platelet-monocyte aggregates. Supernatants from HMEC-1 exposed only to Stx2 exhibited enhanced exposure of platelet P-selectin and platelet-THP-1 cell interactions. Blockade of platelet cyclooxygenase by indomethacin prevented functional activation. The chemokine RANTES enhanced platelet aggregation induced by SDF-1alpha, macrophage-derived chemokine, or thymus and activation-regulated chemokine in the presence of very low ADP levels. These data support the hypothesis that microvascular endothelial cells exposed to E. coli O157:H7-derived Stx2 and LPS release chemokines and other factors, which when combined with low levels of primary agonists, such as ADP, cause platelet activation and promote the renal thrombosis associated with HUS.     

Reconstitution of neurotransmission by determining communication between differentiated PC12 pheochromocytoma and HEL 92.1.7 erythroleukemia cells

Neurotransmitter release was monitored using fura-2-loaded HEL 92.1.7 cells dispersed among differentiated PC12 cells (loaded with another Ca2+ indicator fluo-3) and immobilised using transparent polycarbonate membrane filters with uniform pore size. Depolarisation with K+ caused a rapid rise in Ca2+ concentration in the PC12 cells, followed by a delayed secondary Ca2+ response in simultaneously monitored nearby HEL cells. There was a lag period of about 20 s between the responses of the two cell types. Voltage-gated Ca2+ channels in PC12 cells were inhibited by the P/Q-type (omega-conotoxin MVIIC, omega-agatoxin IVA), N-type (omega-conotoxin GVIA) and L-type channel blockers (nifedipine) as determined using fura-2 or whole-cell patch-clamp recordings. The communication between the cell types on the other hand was sensitive to P/Q- and N-type but not to L-type channel blockers. This suggests that, as in neurons, P/Q- and N-type Ca2+ channels mediate the release of neurotransmitters acting on HEL cells. Theoretically, the procedure employed should be sensitive enough to detect single exocytotic events. Our results demonstrate that a random distribution between effector and target cells is sufficient to allow communication between cells in a manner similar to extrasynaptic transmission.     

Soluble interleukin-2 receptor and metalloproteinase-9 expression in head and neck cancer: prognostic value and analysis of their relationships

In a series of 84 head and neck patients, a statistically significant correlation was observed between high serum soluble interleukin (IL)-2 receptor alpha (sIL-2Ralpha) (P = 0.034) and metalloproteinase-9 (MMP-9) concentrations (P = 0.036) at diagnosis and a shorter survival of these patients. As MMP-9 has been shown to mediate cleavage of IL-2Ralpha (CD25) by preactivated T cells, we looked for a relationship between MMP-9 expression and soluble IL-2Ralpha serum concentrations in these cancer patients. We did not find any correlation between intratumoral expression of MMP-9 or serum MMP-9 concentrations and serum sIL-2Ralpha levels. These results led us to reassess the role of MMP-9 in the release of sIL-2Ralpha. Treatment of Kit225 leukaemic cells with recombinant MMP-9 slightly decreased membrane CD25 expression and was associated with an increased concentration of sIL-2Ralpha in the supernatants. However, using a selective inhibitor of MMP-9 we did not succeed in specifically inhibiting the release of sIL-2Ralpha by the Kit225 cell line or by phytohaemagglutinin (PHA)-activated peripheral blood mononuclear cells. In addition, in a preclinical mouse model, basal serum sIL-2Ralpha concentrations and sIL-2Ralpha production by activated cells were not altered in MMP-9-deficient mice compared to wild-type mice. Interestingly, a broad spectrum metalloproteinase inhibitor inhibited the release of sIL-2Ralpha by PHA-activated peripheral blood mononuclear cells, suggesting that in contrast with current views concerning the major role of MMP-9 in the cleavage of membrane IL-2Ralpha, other proteases are involved in the shedding of sIL-2Ralpha. MMP-9 and sIL-2Ralpha appear therefore as independent prognostic markers in head and neck cancers.     

Prevention of extracellular ADP-induced ATP accumulation of the cultured rat spinal astrocytes via P2Y(1)-mediated inhibition of AMPK

P2Y(1) is probably an important subtype of purinergic receptors (P2Rs) in modulation of the astrocyte activation in spinal cord. The aim of this study was to observe the effect of P2Y(1) receptor on the abnormal energy metabolism of the cultured rat spinal astrocyte induced by extracellular adenosine diphosphate (ADP). The results showed that adenosine triphosphate (ATP) and mitochondrial membrane potential (MMP) in the astrocytes were up-regulated in the presence of ADP, which could be enhanced by MRS2179, a specific antagonist for P2Y(1) receptor. A higher level of expression of the AMP-activated protein kinase (AMPK) was found in the presence of MRS2179 and ADP together than that ADP alone. Blocking of AMPK with Compound C could effectively inhibit the enhancing effect of MRS2179 on ADP-induced astrocyte proliferation and ATP accumulation. Our results suggested that the P2Y(1) receptor mediated inhibition of AMPK may help to prevent the astrocytes from over activation induced by extracellular ADP.     

Airway epithelial cells release eosinophil survival-promoting factors (GM-CSF) after stimulation of proteinase-activated receptor 2

BACKGROUND: Epithelium is considered an active participant in allergic inflammation. Proteinase-activated receptor (PAR) 2 is expressed in a variety of cell types, including epithelial cells, and has been implicated in inflammation. OBJECTIVE: PAR-2-mediated activation of airway epithelial cells induces the release of mediators that could promote eosinophil survival and mediate eosinophil recruitment. METHODS: PAR-2-activating peptides were used to activate the human airway epithelial cell line A549, as well as primary cultures of small airway epithelial cells (SAECs). Human peripheral blood eosinophils were cultured in the presence or absence of epithelial cell supernatants. Survival was assessed by using an Annexin V apoptosis detection kit. GM-CSF and eotaxin were measured by using ELISA. RESULTS: Eosinophils undergo apoptosis in the absence of growth factors. Supernatants from PAR-2-activated A549 epithelial cells increased eosinophil survival. Supernatants from resting SAECs also increased eosinophil survival, but supernatants from PAR-2-activated SAECs showed a greater effect. The effect of PAR-2-activated epithelial cell supernatants on eosinophil survival was completely inhibited by a neutralizing anti-GM-CSF antibody but not an anti-IL-5 antibody. Resting A549 cells did not release any detectable GM-CSF, whereas PAR-2-activated cells released 35 pg/10(6) cells. Resting SAECs released 754.3 pg/10(6) cells of GM-CSF, which was further increased to 1360.5 pg/10(6) cells after PAR-2-mediated activation. Budesonide inhibited this PAR-2 effect. PAR-2-activated epithelial cells also released eotaxin. CONCLUSION: PAR-2-mediated activation of airway epithelial cells induced release of GM-CSF, which promoted eosinophil survival and activation. It also induced release of eotaxin, which could mediate eosinophil recruitment to the airways.     

Possible role of mast cell membrane structures in the enhancement of spleen cell proliferation

The role of mast cells and their mediators in the regulation of immune processes is an area of current interest. In previous studies, we demonstrated that rat peritoneal mast cells and their supernatants enhance the spontaneous and mitogen-induced proliferation of immunocytes. The results of this paper indicate that the enhancing factor in mast cell supernatants is not dependent on mast cell activation or on the release of mediators from within the mast cell. Instead it seems to be due to the passive diffusion of surface structures during mast cell culture. Pretreatment of mast cells with glutaraldehyde did not alter the ability of intact mast cells to enhance spleen cell proliferation. In contrast, mast cells lost their enhancing ability if they were pretreated with trypsin followed by glutaraldehyde fixation. Based on these data, we suggest that protein membrane structures on the mast cell surface are responsible for a contact cooperation between mast cells and spleen cells and that they cause the enhancing effect on spleen cell proliferation. This type of interaction may be responsible for the regulation of immune cell function during local immune reactions.

     

Ionophore A23187 induced reductions in toad urinary bladder epithelial cell oxidative phosphorylation and viability: implications for A23187 related declines in epithelial active transport

The divalent cation ionophore A23187 increased oxygen consumption by isolated epithelial cells from toad urinary bladder, and increase similar to that seen with 2,4-dinitrophenol, a classic uncoupler of mitochondrial oxidative phosphorylation. This respiratory stimulation was not seen in calcium-free incubation media. That this A23187 induced rise in cell oxygen consumption was due to a primary uncoupling action on mitochondrial oxidative phosphorylation rather than secondary to stimulation of cellular transport processes and mediated via increased cellular ADP levels was suggested by the ability of A23187 to release the inhibition of cellular respiration by oligomycin, an inhibitor of the mitochondrial proton ATPase which blocks the stimulation of mitochondrial respiration by ADP. Since active transepithelial ion transport and cellular energy production are closely linked processes, the uncoupling action of A23187 in the presence of extracellular calcium is sufficient to account for an acute decline in active ion transport across epithelia without invoking other calcium-mediated processes. Furthermore, isolated epithelial cells exposed to A23187 for 90 min had greater than 50% loss of viability, as measured by failure of Trypan blue exclusion. The subacute A23187 induced declines in transepithelial transport, therefore, may be secondary to its non-specific effects on cell viability.     

Defective control of Epstein-Barr virus-infected B cell growth in patients with X-linked lymphoproliferative disease.

We studied the cellular function and lymphokine production of T cells from patients with X-linked lymphoproliferative disease (XLP) when activated by the challenge with Epstein-Barr virus (EBV) infection. We used an assay system in which T cells were stimulated with membrane antigens of autologous EBV-infected B lymphoblastoid cell lines (B-LCL) and we examined cellular and humoral factors derived from the stimulated T cells which control the growth of EBV-infected B-LCL. Immunoglobulin secretion from the autologous B-LCL was suppressed with radiosensitive suppressor cells in the patients with XLP. The degree of suppression was correlated with the immunoglobulin levels in the serum of the patients with acquired hypogammaglobulinaemia (P less than 0.05). In addition, T cells from the patients with XLP failed to produce interferon-gamma (IFN-gamma) (P less than 0.001). Moreover, the T cell supernatants from the patients with XLP were less potent to inhibit the B-LCL growth. This diminished inhibition of the B-LCL growth was correlated well with the decreased concentration of IFN-gamma in the T cell supernatants. These findings suggest that suppressor cells may be activated in the patients with the hypogammaglobulinaemia phenotype of XLP, but the frequent development of B cell lymphoma in hypogammaglobulinaemia indicate that immunoglobulin suppression may not exert enough pressure on the in vivo growth of EBV-infected B cells. The defective secretion of IFN-gamma may be, at least partially, responsible for the abnormal cytotoxic T cell and natural killer activities found in the patients with XLP, and may indicate the clinical evaluation about the preventive injection of IFN-gamma against the development of malignant lymphoma.     

Induction of Ly-6A/E expression by murine lymphocytes after in vivo immunization is strictly dependent upon the action of IFN-alpha/beta and/or IFN-gamma

Ly-6A/E is a phosphatidylinositol-linked membrane protein which mediates murine T and B cell signalling. IFN-gamma, IFB-alpha/beta, LPS, and IL-4 have all been reported to induce or upregulate Ly-6A/E by normal lymphocytes. Since no systematic study has addressed the stimulant selectivity of Ly-6A/E expression by murine lymphocytes nor investigated its induction and regulation during primary in vivo immune responses we analyzed in vitro Ly-6A/E expression after murine stimuli and during a number of distinct in vivo immunizations. We show that LPS induces B cell Ly-6A/E in vitro by stimulating the release of IFN-alpha/beta by 'contaminating' adherent cells. In the presence of anti-IFN-gamma + anti-IFN-alpha/beta antibodies, no Ly-6A/E was induced upon addition of multiple cytokines, including IL-4, or mitogenic doses of anti-Ig antibody. Furthermore, IFN-gamma-containing, CD4+ T cell (Th1) supernatants potently induced Ly-6A/E by murine B cells whereas IL-4-containing (Th2) supernatants were either weak or ineffective; anti-IFN-gamma + anti-IFN-alpha/beta inhibited Ly-6A/E induction by both Th1 and Th2 supernatants. Immunization of mice with Brucella abortus or poly (I).poly (C) resulted in induction of Ly-6A/E expression by virtually all B and T cells, whereas injection of G alpha M delta led to peak induction of Ly-6A/E by approximately 50% of both B and T cells. Lymphocytes from mice infected with the nematode parasites Nippostrongylus brasiliensis or Heligmosomoides polygyrus expressed no Ly-6A/E.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of staphylococcal enterotoxin B on rodent mast cells.

Staphylococcal enterotoxin B (SEB) was tested in rodent mast cell cultures for the release of serotonin. Both rat RBL-2H3 mast cells and murine peritoneal cells released serotonin after SEB stimulation in culture. Release of serotonin in RBL-2H3 cells depended on the concentration of SEB; an appreciable release was seen at 50 micrograms/ml. The release of serotonin was not due to cell death. Serotonin release could be enhanced by bradykinin but not by vasoactive intestinal peptide, substance P, lipopolysaccharide from Salmonella typhimurium, the calcium ionophore A23187, acetylcholine, adenosine, 5-hydroxyeicosatetraenoic acid, indomethacin, or phorbol myristate acetate. SEB bound directly to the membrane of RBL-2H3 mast cells, and the SEB-binding site, the presumptive receptor, appeared to be a protein. The SEB receptor could not be capped under membrane-capping conditions, and serotonin release could not be enhanced by attempts to cross-link the receptor. These results suggest that mast cells may be an important cell type involved in SEB toxicosis and that release of serotonin may be enhanced by activation of the kinin-kallikrein system.