IL-2 and IL-5 both induce mu S and J chain mRNA in a clonal B cell line, but differ in their cell-cycle dependency for optimal signaling.

We have found that a neoplastic Lyl+ B cell clone (BCL1-3B3) can be stimulated to secrete IgM by a Th1-derived cytokine, IL-2, and/or by a Th2-derived cytokine, IL-5. At suboptimal concentrations these interleukins acted synergistically to enhance IgM secretion. Both IL-2 and IL-5 induced increases in microseconds and J chain mRNA levels. In the presence of both ILs, increases in microseconds and J chain mRNA were additive and paralleled increases in IgM secretion. Using cells synchronized at the G1/S border with excess thymidine or in early G1 using isoleucine-deficient media, IL-2 and IL-5 differed in their cell-cycle dependency for signal transmission. IL-5 appeared to act preferentially in late G1 of the cell cycle. In contrast, IL-2 stimulated S and G2 phase cells slightly more efficiently than cells in G1 of the cell cycle. Furthermore, a twofold increase in high-affinity IL-2R was observed as the cells entered S phase. The results suggest that although IL-2 and IL-5 can independently and additively induce differentiation of the Lyl+ BCL1-3B3 cells, they differ in their point of action during the cell cycle.     

IL-2 secretion and T cell clonal anergy are induced by distinct biochemical pathways.

In Th1 clones, TCR occupancy together with a costimulatory signal from APC results in IL-2 production. TCR occupancy alone results in unresponsiveness (anergy) to antigenic stimulation, a phenomenon that may be important for self-tolerance in vivo. Inasmuch as inositol phosphate production occurs during the induction of anergy other biochemical signals must be necessary for IL-2 production. Here we assess the role of tyrosine-specific protein kinases using the specific inhibitor, genistein. IL-2 secretion and responsiveness were very dependent on tyrosine-specific protein kinase activation and could be completely blocked under conditions where inositol phosphate generation occurred normally. Although anergy induction could also be blocked by inhibition of tyrosine-specific protein kinase activation this probably occurred indirectly via inhibition of inositol phospholipid hydrolysis. The differential susceptibility of IL-2 secretion and anergy induction to inhibition by genistein indicates that positive and negative outcomes of TCR occupancy may be mediated by distinct biochemical pathways.     

IL-2 induction of IL-1 beta mRNA expression in monocytes. Regulation by agents that block second messenger pathways

We have previously shown that in mixed cultures of PBL incubation with human rIL-2 induces the rapid expression of IL-1 alpha and IL-1 beta mRNA. Because studies have demonstrated that IL-2R can be expressed on the surface of human peripheral blood monocytes, we chose to investigate whether IL-1 beta mRNA could be directly induced in purified human monocytes by treatment with Il-2 and, if so, to analyze the second messenger pathways by which it may be controlled. Human monocytes do not spontaneously express IL-1 beta mRNA, but can express the gene as soon as 1 h after treatment with IL-2. The level of IL-1 beta mRNA induced by IL-2 at 5 h in human monocytes was about one-fourth that induced by LPS. LPS induction of IL-1 beta mRNA in human monocytes can be blocked by either an inhibitor of protein kinase C (PKc) 1-(5-isoquinolinesulfonyl)-2-methylpiperazine or an inhibitor of calcium/calmodulin (CaM) kinase N-(6-aminohexyl) 5-chloro-1-naphthalenesulfonamide, suggesting that both PKc and CaM kinase are involved in transducing signals initiated by LPS. In contrast, IL-2 induction of IL-1 beta mRNA expression is blocked only by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, suggesting that PKc, and not CaM kinase, is activated by IL-2. These data suggest that overlapping but distinct second messenger pathways are involved in the transduction of signals initiated by IL-2 and LPS.     

Cyclin A, cyclin B and stringlike are regulated separately in cell cycle arrested trochoblasts of Patella vulgata embryos

 Trochoblasts are the first cells to differentiate during the development of spiralian embryos. Differentiation is accompanied by a cell division arrest. In embryos of the limpet Patella vulgata, the participation of cell cycle-regulating factors in trochoblast arrest was analysed as a first step to unravel its cause. We determined the cell cycle phase in which the trochoblasts are arrested by analysing the subcellular locations of mitotic cyclins. The results show that the trochoblasts are most likely arrested in the G2 phase. This was supported by measurement of the DNA content in trochoblast nuclei after the last division. Trochoblasts complete their final division at the sixth mitotic cycle. This mitotic cycle resembles the first postblastoderm cell cycle of Drosophila, in which mitotic activity is controlled by expression of the string gene. As failure of string expression results in cell cycle arrest in the G2 phase, negative regulation of a Patella string homolog could be responsible for trochoblast arrest. Although Stl messengers disappeared from trochoblasts during their final division, expression was observed again 20 min later. Messengers remained present in all trochoblasts at low levels during further development. Thus, expression of the stringlike gene allows the cell cycle arrest of these cells, whereas in Drosophila cells arrested in division lack string messengers. Received: 10 February 1997 / Accepted: 23 November 1997     

The levels of leukemia inhibitory factor mRNA in a Schwann cell line are regulated by multiple second messenger pathways

Axotomy of sympathetic and sensory neurons leads to changes in their neuropeptide phenotypes. These changes are mediated in part by the induction of leukemia inhibitory factor (LIF) by nonneuronal cells. In the present study, we identified satellite/Schwann cells as a possible source of the injury-induced LIF. Using a Schwann cell line, SC-1 cells, we examined mechanisms of LIF induction. LIF mRNA levels increased rapidly when the cells were treated with 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, phorbol 12-myristate 13-acetate (PMA), or A23187. Among these reagents, PMA was the most efficacious. Inhibition of protein kinase C (PKC) by GF-1 09203X significantly reduced the PMA-induced LIF mRNA levels. As PKC is known to activate the extracellular signal-regulated kinase (ERK) signaling pathway, the involvement of this pathway in the PMA-stimulated induction of LIF mRNA was examined. Phosphorylation of ERKs was increased following PMA treatment in SC-1 cells. Moreover, inhibition of ERK kinase activity by PD98059 dramatically reduced PMA-stimulated phosphorylation of ERKs and induction of LIF mRNA. These results indicate that LIF mRNA levels can be regulated by ERK activation via stimulation of PKC in Schwann cells.     

The effects of IL-4 and IL-5 on the IgA response by murine Peyer's patch B cell subpopulations

IL-5 has been shown to specifically enhance IgA secretion in LPS-stimulated splenic B cell cultures. Maximum enhancement of IgA in such cultures, however, requires IL-4 in addition to IL-5. Because the Peyer's patches (PP), compared with spleen and lymph nodes, are enriched for precursors of IgA-secreting cells, we tested whether IL-4 and IL-5 would have a more profound effect on IgA secretion by polyclonally stimulated PP cells than spleen cells. The combination of IL-4 and IL-5 causes a comparable enhancement of IgA secretion in both LPS-stimulated PP and splenic B cell cultures. The majority of IgA secreted in LPS-stimulated PP cell cultures is derived from the sIgA- population. Furthermore, the binding high level of peanut agglutinin, germinal center subpopulation of PP cells is essentially nonresponsive to LPS, even in the presence of lymphokines; the majority of secreted IgA in these cultures is derived from the binding low level of peanut agglutinin population. In contrast to LPS-stimulated cultures, PP B cells secrete considerably more IgA than splenic B cells when polyclonally stimulated by a clone of autoreactive T cells in the presence of IL-4 and IL-5. The majority of IgA made by T cell-stimulated PP cell cultures is derived from the sIgA+ population. In these cultures, sIgA- PP cells and spleen cells secrete comparable levels of IgA and other non-IgM isotypes suggesting that sIgA- PP B cells are similar to splenic B cells in their potential to switch to IgA. In T cell-stimulated cultures the majority of IgA as well as of all other isotypes is also derived from the nongerminal center, binding low level of peanut agglutinin population.     

MAPK signaling pathways regulate IL-8 mRNA stability and IL-8 protein expression in cystic fibrosis lung epithelial cell lines

Cystic fibrosis (CF) is characterized by a massive proinflammatory phenotype in the lung, caused by mutations in the CFTR gene. IL-8 and other proinflammatory mediators are elevated in the CF airway, and the immediate mechanism may depend on disease-specific stabilization of IL-8 mRNA in CF lung epithelial cells. MAPK signaling pathways impact directly on IL-8 protein expression in CF cells, and we have hypothesized that the mechanism may also involve stabilization of the IL-8 mRNA. To test this hypothesis, we have examined the effects of pharmacological and molecular inhibitors of p38, and downstream MK2, ERK1/2, and JNK, on stability of IL-8 mRNA in CF lung epithelial cells. We previously showed that tristetraprolin (TTP) was constitutively low in CF and that raising TTP destabilized the IL-8 mRNA. We therefore also tested these effects on CF lung epithelial cells stably expressing TTP. TTP binds to AU-rich elements in the 3'-UTR of the IL-8 mRNA. We find that inhibition of p38 and ERK1/2 reduces the stability of IL-8 mRNA in parental CF cells. However, neither intervention further lowers TTP-dependent destabilization of IL-8 mRNA. By contrast, inhibition of the JNK-2 pathway has no effect on IL-8 mRNA stability in parental CF cell, but rather increases the stability of the message in cells expressing high levels of TTP. However, we find that inhibition of ERK1/2 or p38 leads to suppression of the effect of JNK-2 inhibition on IL-8 mRNA stability. These data thus lend support to our hypothesis that constitutive MAPK signaling and proteasomal activity might also contribute, along with aberrantly lower TTP, to the proinflammatory phenotype in CF lung epithelial cells by increasing IL-8 mRNA stability and IL-8 protein expression.     

IL-2 and GM-CSF are regulated by DNA demethylation during activation of T cells, B cells and macrophages

High-dose intravenous immunoglobulin (IVIG) preparations are currently used for the treatment of autoimmune diseases such as immune thrombocytopenic purpura (ITP). Although the mechanisms of IVIG efficacy remain enigmatic, some clinical and laboratory studies suggest that interaction of the Fc domain of IgG, especially the Fc domain of dimeric IgG, with its receptors (Fc gamma receptors; FcγRs) plays an essential role. In this study, IVIG was dimerized with chemical crosslinkers to augment its therapeutic efficacy. Dimerized IVIG was found to have a much higher affinity for FcγRs than monomeric IVIG. In a mouse ITP model, chemically dimerized IVIG abrogated the decrease in platelet numbers in the blood that was caused by an anti-platelet antibody at a dose that was one tenth of the required dose of IVIG. These results suggest that chemical dimerization of IVIG should greatly improve the efficacy of IVIG therapy of ITP.     

Neutrophil CR3 expression and specific granule exocytosis are controlled by different signal transduction pathways.

Neutrophils express receptors (CR3) for the complement fragment C3bi. CR3 expression can be increased by exposure of the cells to chemotactic factors such as FMLP or to the calcium ionophore A23187. It has been suggested that CR3 moieties are stored in the membrane bounding either the secondary or the tertiary (gelatinase containing) granules. To help define the mechanisms mediating CR3 up-regulation, the effects of several inhibitors upon CR3 expression and secondary granule exocytosis were investigated. Pertussis toxin inhibited FMLP-induced (but not A23187-induced) CR3 expression and exocytosis, indicating that an early step in FMLP-induced CR3 expression is activation of a pertussis toxin-sensitive G protein. However, CR3 expression and exocytosis appeared to be controlled by separate mechanisms distal to G protein activation because 1) DBcAMP and the protein kinase inhibitor H-7 inhibited or stimulated exocytosis, respectively, without affecting CR3 expression; 2) the calmodulin (chlorpromazine and trifluoperazine) and myosin L chain kinase (ML9) inhibitors had greater effects on exocytosis than on CR3 expression; and 3) the kinetics of CR3 expression and exocytosis differed markedly. Thus, although G protein activation is a common early step in both processes, there is a bifurcation of the two processes distally. The mechanisms mediating CR3 up-regulation and tertiary granule exocytosis were also investigated. Extracellular Ca2+ was essential for tertiary granule exocytosis, but not for CR3 up-regulation. We conclude that because the mechanisms controlling CR3 up-regulation and exocytosis diverge soon after the binding of a chemotactic ligand to its receptor, that at least the bulk of increased CR3 expression is not simply a by-product of secondary and tertiary granule exocytosis but is the result of the mobilization of CR3 moieties from an intracellular pool of uncertain identity.     

The amphicrine pancreatic cell line, AR42J, secretes GABA and amylase by separate regulated pathways.

Treatment of AR42J cells with dexamethasone leads to an enhanced formation of amylase-containing granules and facilitates their regulated secretion. Besides the exocrine properties, AR42J cells possess a specific uptake system for [3H]GABA. The stored GABA can be released upon potassium depolarisation in a Ca(2+)-dependent manner. After treatment with dexamethasone, potassium depolarisation fails to release GABA, but instead causes a Ca(2+)-dependent secretion of amylase. Since vesicles similar to small synaptic vesicles of neurons have been identified in AR42J cells, we suggest that the regulated GABA release is mediated by this vesicle type. It is tentatively speculated that other epithelial cells, which also contain small synaptic vesicles and amino acid neurotransmitters, may release them in a similar fashion.     

Both B151-T cell replacing factor 1 and IL-5 regulate Ig secretion and IL-2 receptor expression on a cloned B lymphoma line

In the present study, we have demonstrated that both B151-T cell-replacing factor 1 and rIL-5 are responsible for the activity to partially induce CL-3 cells into IgM-synthesizing cells and also to synergize with IL-2 to augment IL-2R expression on and IgM synthesis in CL-3 cells. These actions of rIL-5 on a homogeneous cloned line (BCL1-CL-3 cells) allow us to identify and characterize the two alternated B cell developmental pathways. One is an IL-2-independent, IL-5-driven differentiation pathway without preceding up-regulated IL-2R expression, and the other is an IL-5 plus IL-2-dependent augmented differentiation pathway with preceding up-regulated IL-2R expression. We have also demonstrated the functional difference of two distinct B cell growth-promoting factors, B cell-stimulating factor 1 (rIL-4) and rIL-5. CL-3 cells are equally stimulated to grow by rIL-4 and rIL-5, whereas only rIL-5 can render CL-3 cells responsive to rIL-2, indicating that these two lymphokines affect B cells in a strikingly different manner.     

IL-7 induces human lymphokine-activated killer cell activity and is regulated by IL-4.

Induction of lymphokine-activated killer (LAK) activity by IL-2 has been described and characterized as broadly cytolytic activity against both fresh and cultured tumors. rIL-7 in the absence of IL-2 also induces LAK activity in human cells. This activity is unique for IL-7, because it is not shared by other cytokines including IL-1, IL-4, IL-6, and TNF-alpha. IL-7 also induces either de novo or increased expression of the surface markers CD25 (Tac, IL-2R alpha-chain), CD54 (ICAM-1), Mic beta 1 (IL-2R beta-chain) and CD69 (early T cell activation Ag). IL-7-induced LAK activity is independent of IL-2 secretion, because it is not abrogated by IL-2 antisera. The LAK precursor responding to IL-7 stimulation is enriched in the null cell fraction as has been demonstrated for IL-2-induced LAK cells. TGF-beta and IL-4 interfere with generation of LAK activity by IL-7. Anti-IL-4 antiserum enhances IL-7-induced LAK activity and augments induction of surface marker expression by IL-7. This may be indirect evidence that IL-7 stimulation leads to induction of IL-4 activity. Our results describe the activation of mature lymphoid cells by IL-7. This and the previously described role of IL-7 in lymphohemopoiesis makes it a cytokine of potential therapeutic value for treatment of immunodeficiency states and possibly the immunotherapy of cancer.     

Tumor cell migration and invasion are regulated by expression of variant integrin glycoforms

The ST6Gal-I glycosyltransferase, which adds α2-6-linked sialic acids to glycoproteins, is overexpressed in colon adenocarcinoma, and enzyme activity is correlated with tumor cell invasiveness. Previously we reported that forced expression of oncogenic ras in HD3 colonocytes causes upregulation of ST6Gal-I, leading to increased α2-6 sialylation of β1 integrins. To determine whether ras-induced sialylation is involved in promoting the tumor cell phenotype, we used shRNA to downregulate ST6Gal-I in ras-expressors, and then monitored integrin-dependent responses. Here we show that forced ST6Gal-I downregulation, leading to diminished α2-6 sialylation of integrins, inhibits cell adhesion to collagen I, a β1 ligand. Correspondingly, collagen binding is reduced by enzymatic removal of cell surface sialic acids from ras-expressors with high ST6Gal-I levels (i.e., no shRNA). Cells with forced ST6Gal-I downregulation also exhibit decreased migration on collagen I and diminished invasion through Matrigel. Importantly, GD25 cells, which lack β1 integrins (and ST6Gal-I), do not demonstrate differential invasiveness when forced to express ST6Gal-I, suggesting that the effects of variant sialylation are mediated specifically by β1 integrins. The observation that cell migration and invasion can be blocked in oncogenic ras-expressing cells by forcing ST6Gal-I downregulation implicates differential sialylation as an important ras effector, and also suggests that ST6Gal-I is a promising therapeutic target.     

Chiral mutagenesis of insulin. Foldability and function are inversely regulated by a stereospecific switch in the B chain

How insulin binds to its receptor is unknown despite decades of investigation. Here, we employ chiral mutagenesis-comparison of corresponding d and l amino acid substitutions in the hormone-to define a structural switch between folding-competent and active conformations. Our strategy is motivated by the T --> R transition, an allosteric feature of zinc-hexamer assembly in which an invariant glycine in the B chain changes conformations. In the classical T state, Gly(B8) lies within a beta-turn and exhibits a positive phi angle (like a d amino acid); in the alternative R state, Gly(B8) is part of an alpha-helix and exhibits a negative phi angle (like an l amino acid). Respective B chain libraries containing mixtures of d or l substitutions at B8 exhibit a stereospecific perturbation of insulin chain combination: l amino acids impede native disulfide pairing, whereas diverse d substitutions are well-tolerated. Strikingly, d substitutions at B8 enhance both synthetic yield and thermodynamic stability but markedly impair biological activity. The NMR structure of such an inactive analogue (as an engineered T-like monomer) is essentially identical to that of native insulin. By contrast, l analogues exhibit impaired folding and stability. Although synthetic yields are very low, such analogues can be highly active. Despite the profound differences between the foldabilities of d and l analogues, crystallization trials suggest that on protein assembly substitutions of either class can be accommodated within classical T or R states. Comparison between such diastereomeric analogues thus implies that the T state represents an inactive but folding-competent conformation. We propose that within folding intermediates the sign of the B8 phi angle exerts kinetic control in a rugged landscape to distinguish between trajectories associated with productive disulfide pairing (positive T-like values) or off-pathway events (negative R-like values). We further propose that the crystallographic T -->R transition in part recapitulates how the conformation of an insulin monomer changes on receptor binding. At the very least the ostensibly unrelated processes of disulfide pairing, allosteric assembly, and receptor binding appear to utilize the same residue as a structural switch; an "ambidextrous" glycine unhindered by the chiral restrictions of the Ramachandran plane. We speculate that this switch operates to protect insulin-and the beta-cell-from protein misfolding.     

Modulation of IgM secretion and H chain mRNA expression in CH12.LX.C4.5F5 B cells by adrenocorticotropic hormone

The murine B cell line CH12.LX.C4.5F5 (CH12 (5F5) expresses adrenocorticotropin (ACTH) receptors, which can modulate IgM secretion by these cells. Interestingly, the response to ACTH was concentration dependent, inducing IgM secretion at subnanomolar amounts and suppressing secretion at micromolar amounts. With the use of an enzyme-linking immunospot assay it was possible to demonstrate that the ACTH-induced increase in IgM secretion by CH12 (5F5) cells was caused at least in part by an increase in the number of cells secreting IgM. CH12 (5F5) cells activated with suboptimal concentrations of LPS demonstrated a similar biphasic response. ACTH at concentrations of 10(-13) to 10(-9) M augmented IgM secretion in LPS-activated cells as much as sixfold, whereas 10(-6) M ACTH slightly decreased LPS-induced IgM secretion. At the mRNA level, subnanomolar concentrations of ACTH increased microH chain mRNA expression up to twofold in unstimulated or LPS-stimulated CH12 (5F5) cells. Taken together, these studies show that physiologically relevant concentrations of ACTH can interact directly with receptors on these B lymphocytes to enhance IgM secretion and microH chain mRNA expression. Although ACTH does increase intracellular cAMP levels in CH12 (5F5) B cells, it is unlikely that the induction of this second messenger pathway is by itself responsible for the ACTH induced B cell differentiation. The concentration of ACTH necessary to stimulate significant intracellular cAMP increases was 10- to 100-fold higher than that required to increase IgM secretion. Furthermore, CH12 (5F5) cells treated with varying concentrations of 8-bromo cAMP or cholera toxin were inhibited in their ability to secrete IgM. These results strongly suggest that the enhancing effects of ACTH on CH12 (5F5) IgM secretion are via mechanisms independent of those mediated by cAMP.     

IL-2 gene expression and NF-kappa B activation through CD28 requires reactive oxygen production by 5-lipoxygenase.

Activation of the CD28 surface receptor provides a major costimulatory signal for T cell activation resulting in enhanced production of interleukin-2 (IL-2) and cell proliferation. In primary T lymphocytes we show that CD28 ligation leads to the rapid intracellular formation of reactive oxygen intermediates (ROIs) which are required for CD28-mediated activation of the NF-kappa B/CD28-responsive complex and IL-2 expression. Delineation of the CD28 signaling cascade was found to involve protein tyrosine kinase activity, followed by the activation of phospholipase A2 and 5-lipoxygenase. Our data suggest that lipoxygenase metabolites activate ROI formation which then induce IL-2 expression via NF-kappa B activation. These findings should be useful for therapeutic strategies and the development of immunosuppressants targeting the CD28 costimulatory pathway.