Effect of GM-CSF on RNA and protein in human peripheral blood granulocytes.

Abstract

Human granulocytes are the most abundant circulating leukocyte, and are critical in protection against microbial infection. These cells are terminally differentiated and highly specialized, possessing very little RNA and protein synthesis. It was believed that transcription and translation were unnecessary for granulocyte functioning, but it is now understood that these processes play a role in some responses. The cytokine GM-CSF, which can be released at inflammatory sites, is an important granulocyte effector. At least one GM-CSF-induced response (i.e., delay in spontaneous apoptosis) has been shown previously to require both transcription and translation. I undertook to examine RNA and protein in these cells, given the importance and need of study. (i) Using DDRT-PCR, hsgk (a putative serine/threonine kinase) mRNA was found to be upregulated ∼10-fold for at least 12 h with ≥ 0.1 ng/mL GM-CSF. (ii) GM-CSF increased [3H]uridine uptake into RNA by ∼10-fold in 6-h cultures. At least 90% of 3 H-RNA was derived from polymerase II, but most appeared to be nonpolyadenylated hnRNA. Granulocytes appear sluggish to synthesize mature cytoplasmic mRNA. Probing granulocyte total RNA with serglycin proteoglycan intron sequences revealed that transcriptional elongation may be the rate-limiting step. (iii) An unknown 26--27 kDa protein demonstrated the largest increase in [ 35S]methionine-labeling (2--4 fold) with GM-CSF-stimulation. This protein could not be identified. (iv) Immunoblotting of Bcl-2 family members revealed only the pro-apoptotic Bax. These proteins apparently are not involved in GM-CSF-induced apoptotic delay. (v) hTegt mRNA was found highly expressed in granulocytes, but was unaffected by GM-CSF. This conserved, little-studied protein was analyzed further. Although the function of hTegt in granulocytes and other cells could not be determined, several properties were uncovered that could be beneficial in future studies. More RNA and protein species remain to be identified in granulocytes, but this work has furthered understanding of the few that are expressed.