High Molecular Weight Basic and Acidic Immunosuppressive Protein Components in Uterine Secretions of Pregnant Cows1

Immunosuppressive proteinaceous components were determined in bovine uterine milk (UTM) collected during late pregnancy. Crude UTM was separated by ion-exchange (carboxymethylcellulose-CMC) and gel filtration (Sephacryl S-200 and Sepharose CL-6B) chromatography. Basic (CMC+) and acidic (CMC-) protein molecular weight (Mr) components were tested for immunosuppressive activity in an in vitro mitogen (phytohemagglutinin-PHA)-treated lymphocyte blastogenesis assay. For most experiments, cultures containing 1 x 10(5) lymphocytes were incubated with 0.08 micrograms PHA and varying concentrations of test protein in RPMI-1640 with supplements. At 48 +/- 1 h, 0.1 microCi of [3H]thymidine was added to cultures and [3H]DNA was quantified at 60 +/- 1 h of culture. Results were expressed as percentage of control values. Crude UTM, CMC+, and CMC- components exhibited immunosuppressive activity. For immunosuppressive Sephacryl S-200 fractions, activity was greater (p less than 0.05-0.01) for CMC+, S-200 fraction I (greater than or equal to 250,000 Mr, void volume [Vo]) than for CMC-, S-200 fractions I (Vo) and III combined for protein concentrations of 20, 40, and 50 micrograms/ml. For the high Mr Sepharose CL-6B protein components, CMC+, CL-6B fraction I (greater than or equal to 4 x 10(6) Mr, Vo) exhibited greater (p less than 0.05-0.005) activity than CMC-, CL-6B fractions I (greater than or equal to 4 x 10(6) Mr, Vo) and II (2.8 x 10(6) Mr) combined at protein concentrations ranging from 20 to 80 micrograms/ml. In summary, bovine UTM contains basic and acidic immunosuppressive protein components, with the greatest activity being associated with a high Mr, basic component.