Identification of wild type and variants of oestrogen receptors in polymorphonuclear and mononuclear leucocytes.
OBJECTIVE
Leucocytes play an important role in the pathogenesis of autoimmune and cardiovascular diseases. Clinical and epidemiological observations indicate that the sex steroid hormones, particularly oestrogens, may regulate leucocyte functions. The assumption that oestrogens have a direct effect on leucocytes has to be supported by identification of functional oestrogen receptors (ER) in leucocytes. This study aimed at investigating the presence of ER subtypes in different types of leucocytes isolated from peripheral blood of female and male donors.


METHODS
A total of nine men (age range 18-43 years) and nine women (age range 19-42 years) all healthy blood donors, were recruited for the study. The donors did not receive any medication or hormonal contraceptives for the last three months. Ten millilitres of peripheral blood was collected from each donor. Polymorphonuclear leucocytes (PMN) and peripheral blood mononuclear cells (PBMC) were purified by density gradient centrifugation.


METHODS
ERalpha and ERbeta mRNA expression was measured by real-time reverse transcriptase-polymerase chain reaction (RT-PCR), and ER proteins were analysed by Western blot in the PBMC and PMN leucocyte populations. In addition, expression profiles of ER variant isoforms were characterized by conventional PCR using the splice-targeted primer approach.


RESULTS
Although we detected wild-type ERalpha and ERbeta mRNAs in PBMC but not in PMN cells, the ERalpha and ERbeta proteins were found in both cell types using Western blot. We observed that both ERalpha and ERbeta proteins differ in size between PMN and PBMC, suggesting that the two leucocyte populations contain diverse variant isoforms of ERalpha and ERbeta. RT-PCR analysis of exon-deleted ER splice variants revealed that PBMC express several exon-deleted variants of ERalpha and ERbeta, along with wild-type receptor, whereas the PMN cells only express exon-deleted variant isoforms and no wild-type ERalpha or ERbeta.


CONCLUSIONS
Our study demonstrates the presence of ERalpha and ERbeta in PBMC and PMN cells from female and male donors. The ERalpha and ERbeta genes have complex transcriptional profiles, with many receptor variant isoforms being expressed. Considering the diversity of ER isoforms in leucocyte subtypes, we conclude that the expected effect of oestrogen would be highly cell type-specific. Further studies are needed to test the functional activity of ER isoforms and their relation to disease.
Stygar
Denis
D
Division for Reproductive Endocrinology, Department of Woman and Child Health, Karolinska Institutet, Stockholm, Sweden. denis.stygar@kbh.ki.se
Westlund
Pär
P
Eriksson
Håkan
H
Sahlin
Lena
L
D000293:Adolescent; D000328:Adult; D000704:Analysis of Variance; D015153:Blotting, Western / Q000379:methods; D047628:Estrogen Receptor alpha / Q000097:blood; D047629:Estrogen Receptor beta / Q000097:blood; D005091:Exons; D005260:Female; D017353:Gene Deletion; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry*; D008183:Luteal Phase / Q000097:blood; D008297:Male; D008970:Molecular Weight; D009504:Neutrophils / Q000737:chemistry*; D020033:Protein Isoforms / Q000032:analysis / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis*; D011960:Receptors, Estrogen / Q000032:analysis / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D020033:Protein Isoforms; D012333:RNA, Messenger; D011960:Receptors, Estrogen