A factor secreted by human embryo stimulates cytokine release by uterine mast cell.
The existence of a biochemical network of embryo-maternal communication implies that various secreted molecules constitute a signal-response mechanism, important for the process of embryo implantation in mammals. Here we report the purification of a protein with an apparent molecular weight of 136 kDa, responsible for a 2000-fold increase in embryo-derived histamine-releasing factor (EHRF) activity. This protein, purified from medium from the in-vitro culture of 2-8-cell human embryos, by means of affinity chromatography, was capable of binding immunoglobulin (Ig)E as demonstrated by immunoblotting and enzyme-linked immunosorbent assays. We found EHRF was capable of inducing release of histamine and cytokines in vitro from rat uterine tissue, collected on day 4 of pregnancy (preimplantation stage of embryo development). When EHRF was used as a secretagogue, granulocyte macrophage-colony stimulating factor (GM-CSF) release increased from 3 to 55 pg/g (P < 0.01) and tumour necrosis factor-alpha (TNF-alpha) release increased from 0 to 2.1 ng/g (P < 0.01), as detected by enzyme-linked immunosorbent assay. A simple method was used to purify uterine mast cells using an IgE-Sepharose affinity chromatography column and the purity (90%) was checked with Dynabeads coupled to specific rat IgE antibody. When purified mast cells were stimulated with EHRF in the same way as the uterine explants, a similar pattern of GM-CSF and TNF-alpha release was obtained. We also describe the reverse transcription-polymerase chain reaction (RT-PCR) of GM-CSF and TNF-alpha mRNA from purified uterine mast cells. On day 4 of pregnancy only the mRNA of TNF-alpha was found and this increased after stimulation with the EHRF. In conclusion, the data presented suggest that uterine mast cells isolated during the preimplantation stage release cytokines in vitro following interaction with an embryo factor.
Cocchiara
R
R
Istituto di Biologia dello Sviluppo, CNR, Palermo, Italia.
Lampiasi
N
N
Albeggiani
G
G
Azzolina
A
A
Bongiovanni
A
A
Gianaroli
L
L
Di Blasi
F
F
Geraci
D
D
D000818:Animals; D001685:Biological Factors / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D017077:Culture Media, Conditioned / Q000737:chemistry; D010064:Embryo Implantation; D004622:Embryo, Mammalian / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005307:Fertilization in Vitro; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism*; D006636:Histamine Release / Q000187:drug effects*; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D008407:Mast Cells / Q000378:metabolism*; D008970:Molecular Weight; D011247:Pregnancy; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D017208:Rats, Wistar; D013268:Stimulation, Chemical; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*; D014599:Uterus / Q000737:chemistry / Q000166:cytology / Q000187:drug effects*
D001685:Biological Factors; D017077:Culture Media, Conditioned; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D007073:Immunoglobulin E; D016178:Granulocyte-Macrophage Colony-Stimulating Factor