Differential regulation of surface Ig- and Lyb2-mediated B cell activation by cyclic AMP. I. Evidence for alternative regulation of signaling through two different receptors linked to phosphatidylinositol hydrolysis in murine B cells.

The differential effect of cAMP on the regulation of early biochemical and cellular functions mediated through two different receptors on murine B cells are reported here. Surface IgM, the Ag receptor, and Lyb2, a 45-kDa differentiation Ag are concomitantly expressed on mature murine B lymphocytes. Triggering of B cells through these molecules, independently, resulted in inositol 1,4,5-triphosphate (IP3) generation, increase in intracellular Ca2+ levels, and cell enlargement associated with progression of cells from G0 to G1 ultimately resulting in DNA synthesis. Pretreatment of resting B cells with cholera toxin as well as other agents that raise the intracellular cAMP [(cAMP)i] such as forskolin, N6,2'-O-dibutyryl cyclic AMP, and 3-isobutyl-1 methyl xanthine inhibited the Ag receptor but not Lyb2-mediated DNA synthesis. The elevation of (cAMP)i inhibited the surface IgM but not Lyb2-mediated IP3 generation, Ca2+ response, and progression from G0 to G1 phase of the cell cycle. Failure of forskolin or N6,2'-O-dibutyryl cyclic AMP to inhibit Lyb2-mediated responses did not appear to be due to induction of cAMP-specific phosphodiesterase activity. Concentrations of H8 [N-(2-(methylamino)-ethyl)-5-isoquinoline sulfonamide, diHCl] inhibitory to cAMP dependent PKA prevented the inhibitory effect of forskolin on surface IgM-mediated Ca2+ response, suggesting that cAMP exerted its effects through PKA. These findings suggest that distinct PLC-coupled receptors, such as sIgM and Lyb2 molecules in B cells, may use either alternative mechanisms for phosphatidylinositol 4,5-bisphosphate hydrolysis or may use different intermediary transducer molecules that differ in their sensitivity to increased (cAMP)i levels. Thus "cross-talk" among cAMP and phosphatidylinositol signaling pathways was demonstrated for IgM but not Lyb2-mediated B cell activation.     

CD47 signals T cell death.

Activation-induced death of T cells regulates immune responses and is considered to involve apoptosis induced by ligation of Fas and TNF receptors. The role of other receptors in signaling T cell death is less clear. In this study we demonstrate that activation of specific epitopes on the Ig variable domain of CD47 rapidly induces apoptosis of T cells. A new mAb, Ad22, to this site induces apoptosis of Jurkat cells and CD3epsilon-stimulated PBMC, as determined by morphological changes, phosphatidylserine exposure on the cell surface, uptake of propidium iodide, and true counts by flow cytometry. In contrast, apoptosis was not observed following culture with anti-CD47 mAbs 2D3 or B6H12 directed to a distant or closely adjacent region, respectively. CD47-mediated cell death was independent of CD3, CD4, CD45, or p56lck involvement as demonstrated by studies with variant Jurkat cell lines deficient in these signaling pathways. However, coligation of CD3epsilon and CD47 enhanced phosphatidylserine externalization on Jurkat cells with functional CD3. Furthermore, normal T cells required preactivation to respond with CD47-induced apoptosis. CD47-mediated cell death appeared to proceed independent of Fas or TNF receptor signaling and did not involve characteristic DNA fragmentation or requirement for IL-1beta-converting enzyme-like proteases or CPP32. Taken together, our data demonstrate that under appropriate conditions, CD47 activation results in very rapid T cell death, apparently mediated by a novel apoptotic pathway. Thus, CD47 may be critically involved in controlling the fate of activated T cells.     

Inhibition of CD3-linked phospholipase C by phorbol ester and by cAMP is associated with decreased phosphotyrosine and increased phosphoserine contents of PLC-gamma 1.

The mechanisms by which phorbol 12-myristate 13-acetate (PMA) and cAMP attenuate the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2) induced by ligation of the T-cell antigen receptor complex (TCR) was studied in the human Jurkat T-cell line. It has previously been shown that stimulation of Jurkat cells with antibodies to CD3, components of the TCR, elicits a rapid and transient phosphorylation of phospholipase C (PLC)-gamma 1, the predominant PLC isozyme in Jurkat cells, at multiple tyrosine residues and that such tyrosine phosphorylation leads to activation of PLC-gamma 1. Prior incubation of Jurkat cells with PMA or forskolin, which increases intracellular cAMP concentrations, prevented tyrosine phosphorylation of PLC-gamma 1 as well as the hydrolysis of PtdIns 4,5-P2 induced by ligation of CD3. Dose-response curves of PMA and of forskolin for the inhibition of PLC-gamma 1 tyrosine phosphorylation and of PtdIns 4,5-P2 hydrolysis were similar. These results suggest that the inhibition of PtdIns 4,5-P2 hydrolysis by PMA and cAMP is attributable to reduced tyrosine phosphorylation of PLC-gamma 1. Treatment of Jurkat cells with PMA or forskolin stimulated the phosphorylation of PLC-gamma 1 at serine 1248. PMA treatment also elicited the phosphorylation of PLC-gamma 1 at an unidentified serine site. Phosphopeptide map analysis indicated that the sites of PLC-gamma 1 phosphorylated in Jurkat cells treated with PMA and forskolin are the same as those phosphorylated in vitro by protein kinase C (PKC) and cAMP-dependent protein kinase (PKA), respectively. Stimulation of Jurkat cells with antibodies to CD3 also elicited phosphorylation of PLC-gamma 1 at serine 1248 and at the unidentified serine site phosphorylated in PLC-gamma 1 from PMA-treated cells. Thus, phosphorylation of PLC-gamma 1 by PKC or PKA at serine 1248 may modulate the interaction of PLC-gamma 1 with the protein tyrosine kinase or the protein tyrosine phosphatase; this altered interaction may, at least in part, be responsible for the decreased tyrosine phosphorylation of PLC-gamma 1 seen in PMA- and forskolin-treated Jurkat cells. Furthermore, in the absence of PMA, activation of PKC by diacylglycerol provides a negative feedback signal responsible for reducing the phosphotyrosine contents of PLC-gamma 1.     

CD47 augments Fas/CD95-mediated apoptosis

Fas (CD95) mediates apoptosis of many cell types, but the susceptibility of cells to killing by Fas ligand and anti-Fas antibodies is highly variable. Jurkat T cells lacking CD47 (integrin-associated protein) are relatively resistant to Fas-mediated death but are efficiently killed by Fas ligand or anti-Fas IgM (CH11) upon expression of CD47. Lack of CD47 impairs events downstream of Fas activation including caspase activation, poly-(ADP-ribose) polymerase cleavage, cytochrome c release from mitochondria, loss of mitochondrial membrane potential, and DNA cleavage. Neither CD47 signaling nor raft association of CD47 is required to enable Fas apoptosis. CH11 induces association of Fas and CD47. Primary T cells from CD47-null mice are also protected from Fas-mediated killing relative to wild type T cells. Thus CD47 associates with Fas upon its activation and augments Fas-mediated apoptosis.     

PGE2-mediated inhibition of T cell p59fyn is independent of cAMP

We recently observed that prostaglandinE₂(PGE₂)-mediated suppression of Tcell functions could result from an attenuation ofp59^fyn protein tyrosine kinaseactivity. The present study evaluated the effects of an adenylatecyclase agonist (forskolin) and antagonist (SQ-22536), as well as thoseof cAMP analogues (dibutyryl cAMP and 8-bromo- cAMP), on T cellp59^fyn kinase activity. The studyallowed us to assess whetherPGE₂-mediated activation ofadenylate cyclase by itself or the elevation in intracellular cAMPlevels is an integral event in the modulation of anti-CD3-linkedp59^fyn activation in T cells. Theexperiments were carried out with splenic T cells from maleSprague-Dawley rats. A 30-50% suppression in theautophosphorylation and the kinase activity ofp59^fyn in T cells incubated withPGE₂ or forskolin was observed.Pretreatment of T cells with SQ-22536 prevented significantPGE₂-mediated inhibition of T cellp59^fyn kinase activity. Incontrast, no change in p59^fynautophosphorylation and kinase activity in T cells treated with cAMPanalogues was observed. These data suggest thatPGE₂-mediated suppression ofp59^fyn autophosphorylation andkinase activity in T cells is dependent on the activation of adenylatecyclase and independent of the elevation in cAMP levels.

     

16,16-dimethyl prostaglandin E2 modulation of endothelial monolayer paracellular barrier function

Prostaglandins prevent gastrointestinal mucosal injury and promote healing following mucosal injury by various noxious agents. Preservation or repair of microvascular function appears to be crucial in these processes. The processes involved in prostaglandin-mediated repair and preservation of endothelial function are unclear. In the present study, we investigated the role of prostaglandins on endothelial paracellular barrier function using the filter-grown bovine aortic endothelial cell monolayers. Endothelial paracellular barrier function was assessed using a paracellular marker, mannitol. Prostaglandin analogs 16,16-dimethyl prostaglandin E₂ (DMPGE₂) and prostaglandin I₂ (PGI₂) caused an enhancement of endothelial monolayer paracellular barrier function as evidenced by a dose-dependent decrease in endothelial paracellular permeability. DMPGE₂ induced enhancement of endothelial paracellular barrier function correlated directly with increasing intracellular cAMP levels. Agents which increase intracellular cAMP levels at different stages of cAMP amplification cascade including phosphodiesterase inhibitor (3-isobutyl-1 methylxanthine [IBMX]), membrane permeable cAMP (8-bromo cAMP), and adenylate cyclase activators (isoproterenol and forskolin) also produced enhancement in endothelial paracellular barrier function. DMPGE₂ enhancement of paracellular barrier function correlated with dense accumulation of actin microfilaments near the intercellular junctions. IBMX, isoproterenol, forskolin, and 8-bromo cAMP also produced similar changes in endothelial actin microfilaments. Cytochalasin B prevented the DMPGE₂ enhancement of paracellular barrier function. Indomethacin (INDO), a cyclooxygenase inhibitor, caused a dose-dependent increase in endothelial paracellular permeability. Pharmacologic doses of INDO resulted in condensation and disruption of actin microfilaments with formation of large paracellular openings or gaps between the adjacent cells. Pretreatment of endothelial monolayers with DMPGE₂ prevented INDO-induced disturbance of actin microfilaments and paracellular barrier function. IBMX, isoproterenol, forskolin, and 8-bromo cAMP also prevented INDO-induced changes in actin microfilaments and paracellular barrier function. These findings indicate that DMPGE₂ has a paracellular barrier enhancing effect on filter-grown endothelial monolayers. This effect appears to be mediated through intracellular cAMP and actin microfilaments. © 1996 Wiley-Liss, Inc.     

CD47(Low) Status on CD4 Effectors Is Necessary for the Contraction/Resolution of the Immune Response in Humans and Mice

How do effector CD4 T cells escape cell death during the contraction of the immune response (IR) remain largely unknown. CD47, through interactions with thrombospondin-1 (TSP-1) and SIRP-α, is implicated in cell death and phagocytosis of malignant cells. Here, we reported a reduction in SIRP-α-Fc binding to effector memory T cells (T(EM)) and in vitro TCR-activated human CD4 T cells that was linked to TSP-1/CD47-induced cell death. The reduced SIRP-α-Fc binding (CD47(low) status) was not detected when CD4 T cells were stained with two anti-CD47 mAbs, which recognize distinct epitopes. In contrast, increased SIRP-α-Fc binding (CD47(high) status) marked central memory T cells (T(CM)) as well as activated CD4 T cells exposed to IL-2, and correlated with resistance to TSP-1/CD47-mediated killing. Auto-aggressive CD4 effectors, which accumulated in lymph nodes and at mucosal sites of patients with Crohn's disease, displayed a CD47(high) status despite a high level of TSP-1 release in colonic tissues. In mice, CD47 (CD47(low) status) was required on antigen (Ag)-specific CD4 effectors for the contraction of the IR in vivo, as significantly lower numbers of Ag-specific CD47(+/+)CD4 T cells were recovered when compared to Ag-specific CD47(-/-) CD4 T cells. In conclusion, we demonstrate that a transient change in the status of CD47, i.e. from CD47(high) to CD47(low), on CD4 effectors regulates the decision-making process that leads to CD47-mediated cell death and contraction of the IR while maintenance of a CD47(high) status on tissue-destructive CD4 effectors prevents the resolution of the inflammatory response.     

Glutathione dependence of caspase-8 activation at the death-inducing signaling complex.

Apoptosis triggered by the death receptor CD95 (APO-1 or Fas) is pivotal for the homeostasis of the immune system. We investigated differential effects of glutathione depletion on CD95-triggered apoptosis in T and B cell lines as well as the glutathione dependence of caspase-8 activation. In B lymphoblastoid SKW6.4 cells, CD95-mediated apoptosis was prevented upstream of caspase-8 activation and caspase-3-like activity after acute glutathione depletion by diethyl maleate or cis-chloro-dinitrobenzene. Immunoprecipitation of the death-inducing signaling complex (DISC) revealed that the DISC was still formed in the glutathione-depleted state. The first cleavage step of procaspase-8 activation at the DISC, however, was inhibited. Accordingly, under cell-free conditions, radiolabeled procaspase-8 was processed at the immunoprecipitated DISC only after the addition of exogenous dithiothreitol or reduced glutathione. We also observed suppression of CD95-mediated apoptosis in glutathione-depleted CEM and H9 cells. Notably, Jurkat cells still died upon CD95 engagement under this condition, displaying incomplete nuclear fragmentation and a partial switch to necrosis; this may be explained by reduced cytochrome c/dATP-mediated caspase activation observed in cytosol from glutathione-depleted Jurkat cytosol. Our data indicate that the activation of caspase-8 at the DISC and hence CD95-mediated apoptosis induction shows a cell-specific requirement for intracellular glutathione.     

Renal cell carcinoma tumors induce T cell apoptosis through receptor-dependent and receptor-independent pathways

Tumors can promote their own progressive growth by inducing T cell apoptosis. Though previous studies suggested that tumor-mediated T cell killing is receptor dependent, we recently showed that tumor gangliosides also participate, a notion consistent with reports indicating that, in some cell types, gangliosides can activate the intrinsic apoptotic pathway by stimulating reactive oxygen species production, cytochrome c release, and caspase-9 activation. In this study, we used normal peripheral blood T cells, as well as caspase-8-, caspase-9-, and Fas-associated death domain protein-deficient Jurkat cells, to assess whether the death ligands and gangliosides overexpressed by the renal cell carcinoma (RCC) cell line SK-RC-45 can independently stimulate T cell apoptosis as a mechanism of immune escape. Anti-FasL Abs and the glycosylceramide synthase inhibitor 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP) each partially inhibited the ability of SK-RC-45 to kill cocultured activated T cells; together, as purified molecules, RCC gangliosides and rFasL induced a more extensive mitochondrial permeability transition and greater levels of apoptosis than either agent alone, equivalent to that induced by the FasL- and ganglioside-expressing RCC line itself. rFasL-mediated apoptosis was completely inhibited in caspase-8- and Fas-associated death domain protein-negative Jurkat cells, though apoptosis induced by purified gangliosides remained intact, findings that correlate with the observed partial inhibition of SK-RC-45-induced apoptosis in the Jurkat lines with defective death receptor signaling. Western blot analysis performed on lysates made from wild-type and mutant Jurkat cells cocultured with SK-RC-45 revealed caspase activation patterns and other biochemical correlates which additionally supported the concept that tumor-associated gangliosides and FasL independently activate the caspase cascade in T cells through the intrinsic and extrinsic pathways, respectively.     

Protein kinase C inhibits CD95 (Fas/APO-1)-mediated apoptosis by at least two different mechanisms in Jurkat T cells.

We have recently reported that activation of protein kinase C (PKC) plays a negative role in CD95-mediated apoptosis in human T cell lines. Here we present data indicating that although the PKC-induced mitogen-activated protein kinase pathway could be partially implicated in the abrogation of CD95-mediated apoptosis by phorbol esters in Jurkat T cells, the major inhibitory effect is exerted through a PKC-dependent, mitogen-activated protein kinase-independent signaling pathway. Furthermore, we demonstrate that activation of PKC diminishes CD95 receptor aggregation elicited by agonistic CD95 Abs. On the other hand, it has been reported that UV radiation-induced apoptosis is mediated at least in part by the induction of CD95 oligomerization at the cell surface. Here we show that activation of PKC also inhibits UVB light-induced CD95 aggregation and apoptosis in Jurkat T cells. These results reveal a novel mechanism by which T cells may restrain their sensitivity to CD95-induced cell death through PKC-mediated regulation of CD95 receptor oligomerization at the cell membrane.     

Cyclic AMP modulation of human B cell proliferative responses: role of cAMP-dependent protein kinases in enhancing B cell responses to phorbol diesters and ionomycin.

The ability of cyclic AMP (cAMP) to modulate human B cell proliferative responses and the possible role of cAMP-dependent kinases (PKA) in cAMP modulation of proliferative responses were investigated. The addition of dibutyl cAMP (Bt2 cAMP) or the cAMP-elevating agent forskolin to B cells stimulated by crosslinking surface immunoglobulins (sIg) resulted in a concentration-dependent inhibition of proliferative responses. By contrast, Bt2 cAMP or forskolin enhanced the proliferative responses of B cells after direct stimulation by phorbol myristate acetate (PMA) and the calcium ionophore ionomycin. The inhibition and enhancement of B cell proliferative responses by Bt2 cAMP were observed at different incubation intervals and were not due to temporal shifts of optimal responses. Also, Bt2 cAMP caused only small changes in B cell RNA synthesis compared to modulation of proliferative responses. Exposure of B cells to Bt2 cAMP rapidly activated PKA. Blocking Bt2 cAMP activation of PKA with the kinase inhibitor HA1004 prevented Bt2 cAMP enhancement of B cell responses after direct stimulation by PMA and ionomycin. In reciprocal experiments, the kinase inhibitor H7 resulted in some inhibition of PKC activation but did not inhibit Bt2 cAMP activation of PKA or Bt2 cAMP enhancement of proliferative responses. Other experiments demonstrated that B cells treated with Bt2 cAMP had selective increases in the de novo phosphorylations of two endogenous substrates which reflected PKA activation. Furthermore, concentrations of HA1004 or H8 which inhibited Bt2 cAMP enhancement of proliferative responses also inhibited PKA phosphorylations of these substrates whereas H7 did not. Thus, elevations of cAMP can enhance or inhibit human B cell proliferative responses to different stimuli and the activation of PKA is important for cAMP enhancement of certain responses.     

Inhibition of antigen-specific T cell proliferation and cytokine production by protein kinase A type I

cAMP inhibits biochemical events leading to T cell activation by triggering of an inhibitory protein kinase A (PKA)-C-terminal Src kinase pathway assembled in lipid rafts. In this study, we demonstrate that activation of PKA type I by Sp-8-bromo-cAMPS (a cAMP agonist) has profound inhibitory effects on Ag-specific immune responses in peripheral effector T cells. Activation of PKA type I inhibits both cytokine production and proliferative responses in both CD4(+) and CD8(+) T cells in a concentration-dependent manner. The observed effects of cAMP appeared to occur endogenously in T cells and were not dependent on APC. The inhibition of responses was not due to apoptosis of specific T cells and was reversible by a PKA type I-selective cAMP antagonist. This supports the notion of PKA type I as a key enzyme in the negative regulation of immune responses and a potential target for inhibiting autoreactive T cells.     

cAMP-dependent protein kinase is essential for hypoxia-mediated epithelial–mesenchymal transition,migration, and invasion in lung cancer cells

Lung cancer is the leading cause of cancer-related death worldwide. Hypoxia is known to increase cancer cell migration and invasion. We have previously reported that hypoxia induces epithelial–mesenchymal transition (EMT) in lung cancer cells. However, it is unknown whether hypoxia promotes lung cancer cell migration and invasion via EMT and whether cyclic AMP (cAMP) dependent protein kinase (PKA) plays a role in this process. We found that hypoxia increased PKA activity and induced mRNA and protein expression of PKA catalytic subunit α (PKACA), and regulatory subunits R1A and R1B. Knockdown of HIF-1/2α prevented hypoxia-mediated induction of PKACA mRNA expression and PKA activity. Inhibition of PKA activity with chemical inhibitors prevented EMT induced by hypoxia and tumor growth factor β1. However, activation of PKA by forskolin and 8-Br-cAMP did not induce EMT. Furthermore, treatment with H89 and knockdown of PKACA prevented hypoxia-mediated, EMT, cell migration, and invasion, whereas overexpression of mouse PKACA rescued hypoxia-mediated migration and invasion in PKACA deficient cancer cells. Our results suggest that hypoxia enhances PKA activity by upregulating PKA gene expression in a HIF dependent mechanism and that PKA plays a key role in hypoxia-mediated EMT, migration, and invasion in lung cancer cells.     

Induction of polyclonal CD8+ T cell activation and effector function by Pertussis toxin

Pertussis toxin (PTX) has pronounced adjuvant activity and strongly enhances innate and adaptive immune responses, including increased antibody production and Th1/Th2 cytokine production. Adjuvant effects of PTX on Th1 and Th2 cells are primarily mediated via CD80/86 costimulation via enhanced expression of these molecules by APCs. However, it has remained unresolved whether PTX modulates the expression of costimulatory and inhibitory molecules on CD4+ and CD8+ T cells. To address this question, we determined the expression kinetics of CD28, CTLA-4, and CD40L on spleen CD4+ and CD8+ T cells after incubation with PTX. The results show that PTX upregulated the expression of CD28 by CD8+ T cells, but not by CD4+ T cells. In contrast, the expression of CTLA-4 and CD40L was not substantially altered on CD4+ or CD8+ T cells. CD28 upregulation by CD8+ T cells was paralleled by upregulation of CD69 and the induction of IFN-γ, Granzyme B (GrB), and IL-17. CD8+ T cell activation and cytokine production could be substantially blocked with anti-CD80 and CD86 antibodies, consistent with CD28 mediated signaling. Treatment of highly purified CD8+ T cells with PTX resulted in upregulation of CD28 and CD69, and production of IFN-γ. Incubation with CD28 mAb further enhanced this effect, suggesting that PTX has direct effects on CD8+ T cells which are enhanced by CD80/86-mediated costimulation provided by APCs.     

Intracellular mediators regulate CD2 lateral diffusion and cytoplasmic Ca2+ mobilization upon CD2-mediated T cell activation.

CD2 is a T cell surface glycoprotein that participates in T cell adhesion and activation. These processes are dynamically interrelated, in that T cell activation regulates the strength of CD2-mediated T cell adhesion. The lateral redistribution of CD2 and its ligand CD58 (LFA-3) in T cell and target membranes, respectively, has also been shown to affect cellular adhesion strength. We have used the fluorescence photobleaching recovery technique to measure the lateral mobility of CD2 in plasma membranes of resting and activated Jurkat T leukemia cells. CD2-mediated T cell activation caused lateral immobilization of 90% of cell surface CD2 molecules. Depleting cells of cytoplasmic Ca2+, loading cells with dibutyric cAMP, and disrupting cellular microfilaments each partially reversed the effect of CD2-mediated activation on the lateral mobility of CD2. These intracellular mediators apparently influence the same signal transduction pathways, because the effects of the mediators on CD2 lateral mobility were not additive. In separate experiments, activation-associated cytoplasmic Ca2+ mobilization was found to require microfilament integrity and to be negatively regulated by cAMP. By directly or indirectly controlling CD2 lateral diffusion and cell surface distribution, cytoplasmic Ca2+ mobilization may have an important regulatory role in CD2 mediated T cell adhesion.