Unmethylated CpG dinucleotides are more frequent in the genomes of bacteria and viruses than of vertebrates. We report here that bacterial DNA and synthetic oligodeoxynucleotides containing unmethylated CpG dinucleotides induce murine B cells to proliferate and secrete immunoglobulin in vitro and in vivo. This activation is enhanced by simultaneous signals delivered through the antigen receptor. Optimal B-cell activation requires a DNA motif in which an unmethylated CpG dinucleotide is flanked by two 5' purines and two 3' pyrimidines. Oligodeoxynucleotides containing this CpG motif induce more than 95% of all spleen B cells to enter the cell cycle. These data suggest a possible evolutionary link between immune defence based on the recognition of microbial DNA and the phenomenon of 'CpG suppression' in vertebrates. The potent immune activation by CpG oligonucleotides has implications for the design and interpretation of studies using 'antisense' oligonucleotides and points to possible new applications as adjuvants.

Although the dynamics of oscillations of cytosolic Ca2+ concentration ([Ca2+]cyt) play important roles in early mammalian development, the impact of the duration when [Ca2+]cyt is elevated is not known. To determine the sensitivity of fertilization-associated responses [i.e., cortical granule exocytosis, resumption of the cell cycle, Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, recruitment of maternal mRNAs] and developmental competence of the parthenotes to the duration of a [Ca2+]cyt transient, unfertilized mouse eggs were subjected to a prolonged [Ca2+]cyt change for 15, 25, or 50 min by means of repetitive Ca2+ electropermeabilization at 2-min intervals. The initiation and completion of fertilization-associated responses are correlated with the duration of time in which the [Ca2+]cyt is elevated, with the exception that autonomous CaMKII activity is down-regulated with prolonged elevated [Ca2+]cyt. Activated eggs from 25- or 50-min treatments readily develop to the blastocyst stage with no sign of apoptosis or necrosis and some implant. Ca2+ influx into unfertilized eggs causes neither Ca2+ release from intracellular stores nor rapid removal of cytosolic Ca2+. Thus, the total Ca2+ signal input appears to be an important regulatory parameter that ensures completion of fertilization-associated events and oocytes have a surprising degree of tolerance for a prolonged change in [Ca2+]cyt.

The effects of phosphorothioate (S-oligonucleotide) or terminal phosphorothioate-phosphodiester (S-O-oligonucleotides) or methylphosphonate-phosphodiester (MP-O-oligonucleotides) modifications on mouse spleen cell surface binding, uptake, and degradation were studied using fluorescein (FITC)-conjugated oligonucleotides. S-oligonucleotides had the highest cell binding and uptake, followed by S-O-, O-, and MP-O-oligonucleotides. Competition studies indicated that S-oligonucleotides have an increased affinity for cell membrane oligonucleotide binding sites, because they could completely block O-oligonucleotide binding at a molar ratio of just 0.1. Uptake of all oligonucleotides was higher in B cells than T cells and was increased by stimulation with the B-cell mitogen, lipopolysaccharide. Although our cells had been purified using conventional techniques to eliminate dead cells, there remained about 5% of cells that were dead or dying, as determined by flow cytometry using propidium iodide staining. Of note, oligonucleotide association with dead cells was approximately 50-fold greater than that with living cells. Confocal microscopy confirmed that the oligonucleotides in living cells were intracellular, and indicated little nuclear uptake by 4 h. While extensive degradation of intracellular O-oligonucleotides was apparent by 4 h, there was no detectable degradation of S-, S-O, or MP-O-oligonucleotides.

Phosphodiester oligodeoxynucleotides bearing a 5' cholesteryl (chol) modification bind to low density lipoprotein (LDL), apparently by partitioning the chol-modified oligonucleotides into the lipid layer. Both HL60 cells and primary mouse spleen T and B cells incubated with fluorescently labeled chol-modified oligonucleotide showed substantially increased cellular association by flow cytometry and increased internalization by confocal microscopy compared to an identical molecule not bearing the chol group. Cellular internalization of chol-modified oligonucleotide occurred at least partially through the LDL receptor; it was increased in mouse spleen cells by cell culture in lipoprotein-deficient medium and/or lovastatin, and it was decreased by culture in high serum medium. To determine whether chol-modified oligonucleotides are more potent antisense agents, we titered antisense unmodified phosphodiester and chol-modified oligonucleotides targeted against a mouse immunosuppressive protein. Murine spleen cells cultured with 20 microM phosphodiester antisense oligonucleotides had a 2-fold increase in RNA synthesis, indicating the expected lymphocyte activation. Antisense chol-modified oligonucleotides showed an 8-fold increase in relative potency: they caused a 2-fold increase in RNA synthesis at just 2.5 microM. The increased efficacy was blocked by heparin and was further increased by cell culture in 1% (vs. 10%) fetal bovine serum, suggesting that the effect may, at least in part, be mediated via the LDL receptor. Antisense chol-modified oligonucleotides are sequence specific and have increased potency as compared to unmodified oligonucleotides.

Elucidation of the biochemical mechanisms by which specific proteins transduce the all important intracellular calcium (Ca2+) signal at fertilization into events of egg activation will increase our understanding of the regulation of the onset of development and the extent to which these signals can be experimentally modified. Previously, we reported data supporting the hypothesis that mouse eggs have the capability to generate oscillations of the activity of Ca2+ and calmodulin-dependent kinase II (CaMKII), regulating the cell cycle and secretion. This study directly demonstrates transient increases of enzyme activity in relatively close synchrony with Ca2+ oscillations for the first hour of fertilization in single mouse eggs monitored for both Ca2+ and CaMKII activity. The extent of the enzyme activity increase was correlated with the level of intracellular Ca2+. After a rise in activity, the decrease in activity did not appear to be due to negative feedback from elevated Ca2+ or CaMKII activity over time, since enzyme activity persisted after 8 min of elevated Ca2+ from 7% ethanol activation. The contribution of CaMKII from a single sperm to the rise in CaMKII activity at fertilization appeared to be negligible. Also, long-term cell cycle inhibition was observed in fertilized eggs with the CaMKII antagonist myrAIP (50 microM), which did not inhibit the first large Ca2+ transient or subsequent early oscillations but did reduce the percentage of eggs fertilized. Thus, mammalian eggs appear to drive many activation events over time to completion with repeated short bursts of Ca2+ oscillation-dependent CaMKII activity, rather than by a steady-state, continuously elevated level of CaMKII activity that is maintained by periodic Ca2+ oscillations.