Characterization of human lymphocyte derived chemotactic factors for mononuclear phagocytes--II. Chemical...

Supernatants of PHA-stimulated human mononuclear cells were fractionated by Sephadex G-100 chromatography and isoelectric focusing. Chemotactic activities for human mononuclear phagocytes were identified at various molecular weight ranges. Major activities were found at mol. wt 45, 30, 25, 18, 12.5, 8 and 6 K. After isoelectric focusing chemotactic activities were recovered predominantly at pH 3.7-5.5 and 7.8-8.5. Fractionation by high performance liquid chromatography (HPLC) on the molecular sieve column I-125 and basic ion exchange column SAX 300 confirmed these results. Furthermore, after absorption of crude supernatants on the reversed-phase column RP 300, chemotactic activity was recovered quantitatively and thus could be separated from the bulk of other materials. In kinetic experiments supernatants were harvested after 5, 27, 42 and 63 hr and fractionated on a molecular sieve column. It was found that only low molecular weight chemotactic activity was released after 5 hr. After 27 hr most activity was found between 10-20,000 after 42 hr activity was found over the whole fractionation range. It is concluded that multiple molecular species of chemotactic factors acting on mononuclear phagocytes are released by activated lymphocyte cultures, whose chemical nature and function remains to be studied.