Although both GM-CSF and G-CSF activate p42/44 MAPK in neutrophil progenitors, the ability of G-CSF to cause MAPK activation is lost in mature neutrophils, while GM-CSF exposure still causes activation. The mechanism of this differential effect related to maturation status has not been explored. We verified that G-CSF and GM-CSF receptors remain functional on purified mature neutrophils by demonstrating that both cytokines caused phosphorylation of STAT3. However, only GM-CSF was capable of activating MAPK as assessed by gel shift and in vitro kinase assay. Both G-CSF and GM-CSF caused activation of p21 ras in neutrophils, demonstrating that early events in the ras-MAPK pathway remain functional after stimulation by either cytokine. Inhibition of tyrosine phosphatase activity by pervanadate restored the ability of G-CSF to activate MAPK in mature neutrophils. Specific inhibition of the SHP-1 phosphatase, known to be activated by G-CSF but not GM-CSF also restored the ability of G-CSF to activate MAPK in neutrophils. These studies suggest that G-CSF activation of SHP-1 may be an important regulatory step for permitting optimal terminal differentiation during neutrophil production and add to our knowledge of the instructional role of G-CSF and GM-CSF for balancing proliferation and differentiation of neutrophil progenitor cells. This information may prove useful for the understanding of conditions in which neutrophil proliferative/differentiative balancing is dysregulated, such as myeloid leukemia and myelodysplastic disorders.

Antiviral effects of a DNA vaccine against herpes simplex virus 1 (HSV-1) glycoprotein D (gD) were evaluated in eight week-old female BALB/c mice. The nuclease-insensitive construct (gD-ASOR) consisted of an HSV-1 gD encoding plasmid coupled to asialo orosomucoid (ASOR), targeting it to cells bearing ASOR receptors. Mice were immunized on day 0 and 7 with 10 microg doses of gD-ASOR or control substances. Fourteen days later, mice were infected by the corneal route with 10(5) pfu or 10(6) pfu HSV-1, strain 17syn+. Immunized mice showed a significant decrease in ocular disease severity over a 21-day observation period following infection compared to sham-immunized mice. Acute replication kinetic assays demonstrated a 100-fold decrease in viral titers on day 6 in trigeminal ganglia from immunized BALB/c mice compared to sham-immunized mice. Immunized mice showed a significant increase in numbers of CD4(+)T cells infiltrating the trigeminal ganglia at day 6 post infection compared to sham-immunized mice. Significant differences were not seen in latent viral reservoir between immunized and unimmunized mouse groups. Immunization with gD-ASOR decreased the severity of acute ocular HSV-1 infection, induced a CD4(+) T cell response, decreased the viral load in the trigeminal ganglia, but did not diminish viral latency.

The role of ribosomal p70S6K in the cell cycle has been studied extensively, and it is known that this enzyme is crucial for cell advancement through G(1). Conversely, the participation of p70S6K in cell differentiation is not well understood. We have studied the response of p70S6K to the cytokine granulocyte-macrophage colony stimulating factor (GM-CSF) in three differentiation-capable leukemic cell lines (MPD, AML-14 and HL-60) and in normal mature neutrophils. Immature leukemic cells starved for 16 h showed a robust ( approximately 3.5-fold over controls) p70S6K phosphorylation on T(421)/S(424) residues in response to an acute (5 min) 10 nM GM-CSF stimulation. On the other hand, cells that had been induced to differentiate and express granulocytic phenotypes, showed an increased ( approximately 6-fold) basal level of p70S6K T(421)/S(424) phosphorylation over immature cells, as well as an increased baseline tyrosyl phosphorylation of the GM-CSF receptor beta subunit (GM-CSF.Rbeta). However, the differentiated cells displayed a weak ( approximately 1.4-fold over controls) response to GM-CSF even at prolonged incubation times (20 min). In vitro p70S6K enzymatic activity paralleled p70S6K T(421)/S(424) phosphorylation in both high basal, unstimulated, levels in immature cells and a low degree of response to GM-CSF. Lastly, peripheral blood mature neutrophils had low basal GM-CSF.Rbeta and p70S6K activity, with both parameters being robustly stimulated following addition of GM-CSF, a situation in contrast with the cell lines, indicative perhaps of their incomplete terminal differentiation. In summary, these findings show the increase in basal phosphorylation of p70S6K upon granulocytic differentiation of myeloid leukemic cells and their responses to GM-CSF that are closely paralleled with tyrosyl phosphorylation of its receptor, and help in pointing to specific cell signaling molecules that are different in leukemic blasts from normal leukocytes.