Lori Manzel

Structure-activity relationship analysis of substituted 4-quinolinamines, antagonists of immunostimulatory CpG-oligodeoxynucleotides

On the basis of a systematic SAR analysis of substituted quinolines, a derivative 32 was synthesized that shows half-maximal inhibition of the immunostimulatory effect of CpG-oligodeoxynucleotides in vitro at the concentration of 0.24 nM.

Bis-4-aminoquinolines: novel triple-helix DNA intercalators and antagonists of immunostimulatory CpG-oligodeoxynucleotides

Six dimeric 2-(2-naphthyl)quinolin-4-amines with a linker between the amino groups and eight dimeric 2-(4-anilino)quinolin-4-amines linked between the anilino groups were synthesized and evaluated for their interaction with duplex/triplex DNAs and as antagonists of immunostimulatory oligodeoxynucleotides with a CpG-motif (CpG-ODN). The most powerful triple-helix DNA intercalator known to date, with high affinity toward T.A.T triplets and triplex/duplex selectivity, was found. The potent antagonism of immunostimulatory CpG-ODN by several bis-4-aminoquinolines is not related to their DNA interactions.

CpG-oligodeoxynucleotide-resistant variant of WEHI 231 cells

Bacterial DNA and synthetic single‐stranded oligonucleotides having unmethylated CpG motifs (CpG‐ODN) powerfully stimulate cellular immune responses by an unknown mechanism. There is evidence that internalization of the nucleotide is required for activity. Both CpG‐ODN and engagement of CD40 protects WEHI‐231 murine B lymphoma cells from apoptosis induced by antibody to surface IgM, and both agents induce interleukin‐6 (IL‐6) production by these cells. We now report the isolation of CpG‐ODN‐resistant subclones (designated CR) from WEHI 231 cells, as well as subclones that are sensitive to CpG‐ODN (designated CS). CR clones completely fail to respond to CpG‐ODN but they retain the capacity to respond normally to engagement of CD40. CR cells incorporate CpG‐ODN into small, acidified perinuclear vesicles in the same way as do the parent WEHI 231 cells. The CR, CS, and WEHI 231 cells all had identical cytogenetics. The described CR clones have a stable and specific defect in the mechanism responsible for the intracellular recognition and response to CpG‐ODN, suggesting that they harbor a mutation that disables the CpG‐ODN detection mechanism. These clones may be useful to determine at a molecular level which proteins and cell components are required for immune cells to detect and respond to CpG‐ODN. J. Leukoc. Biol. 66: 817–821; 1999.

Protein kinase C is not necessary for transforming growth factor beta-induced growth-arrest in leukemia cell lines

Growth and differentiation of blood cell precursors are regulated by cytokines and hormones by mechanisms which are incompletely understood. Protein kinase C (PKC) isozymes are widely regarded as being important in signal transduction pathways. We have shown that one isozyme, PKC beta, is uniquely important in mediating phorbol ester-induced growth-arrest in the HL-60 myeloid cell line. 1,25-dihydroxyvitamin D3 induces differentiation and growth-arrest in many cells. It upregulates the expression of PKC beta, potentiating the action of phorbol ester. We tested the hypotheses that cytokines, which arrest the growth of hematopoietic cells, do so by activating PKC beta, and that differentiation and growth-arrest induced by 1,25-dihydroxyvitamin D3 is caused by upregulation of PKC beta isozyme gene expression. The influence on growth of combinations of five cytokines (TNF alpha, TGF beta 1, gamma-IFN, IL-1, and G-CSF) and 1,25-dihydroxyvitamin D3 on ten human leukemia cell lines (THP-1, HL-60 S, HL-60 PET, U937, K562, Jurkat, MOLT-4, RPM1 8402, KG-1, and KG-1a) was determined. Four cell lines (THP-1, HL-60 S and PET, and U937) exhibited total growth-arrest when incubated with 1,25-dihydroxyvitamin D3 followed by TGF beta 1. The expression by each cell line of mRNA encoding PKC alpha, beta, and delta, both before and after 24 or 48 h of incubation with 1,25-dihydroxyvitamin D3, was determined. Cell lines sensitive to TGF beta 1 each expressed PKC delta endogenously, or expression was up-regulated with 1,25-dihydroxyvitamin D3. U937 cells underexpressed PKC alpha, and HL-60 PET cells underexpressed PKC beta. These data suggested that PKC delta could be responsible for mediating growth-arrest by TGF beta 1. To test this hypothesis directly, we incubated the cells with two bisindolylmaleimide PKC inhibitors during the addition of 1,25-dihydroxyvitamin D3 and TGF beta 1. Surprisingly, the PKC inhibitors did not block the growth-arrest induced by 1,25-dihydroxyvitamin D3 and TGF beta 1. This experiment strongly suggests that neither growth-arrest induced by TGF beta 1 nor the potentiation of this growth-arrest by 1,25-dihydroxyvitamin D3 is mediated by a PKC isozyme which is inhibitable by the bisindolymaleimides.

Immunostimulatory CpG-oligodeoxynucleotides induce a factor that inhibits macrophage adhesion☆☆☆★

Selected phosphorothioate oligodeoxynucleotides containing CpG (CpG-ODN) activate immune responses, including immunoglobulin synthesis, B cell proliferation, and cytokine production by monocytes. We examined the effect of a CpG-ODN (#1760) on the adhesion of macrophages derived from human blood monocytes in vitro. CpG-ODN (6 microg/mL) completely inhibited the adherence of macrophages to plastic or glass during 7 or more days of culture. A non-CpG control ODN (#1814) was without effect. Two other CpG-ODNs (#1826 and #1842) also completely inhibited macrophage adherence. The specific inhibitor of CpG-ODN, quinacrine (0.1 micromol/L), blocked this action. CpG-ODN reduced the rate of senescence and cell death of monocytes in culture but did not influence their phagocytosis, procoagulant activity, or support of the mixed lymphocyte response. Four days of exposure of monocytes to CpG-ODN up-regulated the expression of the endotoxin receptor CD14 and down-regulated the mannose (scavenger) receptor, a result that is consistent with blocking the maturation of monocytes to macrophages. Incubation of peripheral blood monocytes (PBMCs) with CpG-ODN resulted in the generation of a heat labile factor that inhibited macrophage differentiation and accounts for the efficacy of the CpG-ODN. T cells selected from PBMCs by magnetic beads generated the majority of this factor. Cytokines (interleukin-3 (IL-3), IL-4, IL-6, IL-10, interferon-gamma, tumor necrosis factor-alpha, transforming growth factor-beta, granulocyte-macrophage colony-stimulating factor, monocyte chemotactic protein-1) did not inhibit macrophage adherence like CpG-ODN did. Antibodies to IL-6 or IL-10 did not block the activity of CpG-ODN. Dexamethasone inhibited macrophage adherence, and lipopolysaccharide had a minor effect. We conclude that immunostimulatory CpG-ODNs inhibit macrophage adherence by provoking the production of an unidentified heat-labile factor.