Abstract
Human leukocytes stimulated in vitro release leukocytic pyrogen (LP), a protein which is the mediator of fever. In order to study human leukocytic pyrogen, we attempted to purify this molecule from the large quantity and variety of proteins which are present in leukocyte supernates. Human peripheral leukocytes were stimulated in vitro by phagocytosis of killed staphylococci, and several methods were used to isolate the pyrogen protein. First, using isoelectric focusing, it was found that crude leukocyte supernates contained two molecular species of LP which were separable by precipitation in cold alcohol. Isoelectric focusing, although used for confirmation of the molecular homogeneity of LP, could not be employed as a preparative purification technique. Following alcohol precipitation, human LP was chromatographed on ion-exchange materials at various pH with modest recovery of initial activity but marked increase in specific activity. Gel-filtration was also employed and yielded partially purified LP. When alcohol precipitation was combined with ion exchange at alkaline pH and followed by gel-filtration, resulting LP preparations contained 5 or 6 contaminating proteins. These results demonstrate that human LP can be partially purified from the large quantity and variety of proteins present in crude leukocyte supernates and during purification procedures, the pyrogen did not change in either molecular weight or isoelectric point. This work provides reliable techniques for initial purification of human LP.
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References
Kampschmidt, R.F., H.F. Upchurch, C.L. Eddington, andL.A. Pulliam. 1973. Multiple biological activities of a partially purified leukocytic endogenous mediator.Am. J. Physiol. 244:530.
Kampschmidt, R.F., R.D. Long, andH.F. Upchurch. 1972. Neutrophil releasing activity in rats injected with endogenous pyrogen.Proc. Soc. Exp. Biol. Med. 139:1224.
Kampschmidt, R.F., andH.F. Upchurch. 1974. Effect of leukocytic endogenous mediator on plasma fibrinogen and haptoglobin.Proc. Soc. Exp. Biol. Med. 146:904.
Kampschmidt, R.F., andL.A. Pulliam. 1975. Stimulation of antimicrobial activity in the rat with leukocytic endogenous mediator.J. Reticulo. Soc. 17:162.
Kiley, R.S., andG.W. Rafter. 1975. Activation of rabbit brain microsomal (Na+K)-dependent ATPase by a leukocytic product.Biochem. Biophys. Acta. 381:120.
Atkins, E., andP. Bodel. 1972. Fever.N. Engl. J. Med. 286:27.
Dinarello, C.A., N.P. Goldin, andS.M. Wolff. 1974. Demonstration and characterization of two distinct human leukocytic pyrogens.J. Exp. Med. 139:1369.
Perlow, M., C.A. Dinarello, andS.M. Wolff. 1975. A primate model for the study of human fever.J. Infect. Dis. 132:157.
Bodel, P.T., A. Wechsler, andE. Atkins. 1969. Comparison of endogenous pyrogens from human and rabbit leukocytes utilizing Sephadex filtration.Yale J. Biol. Med. 41:376.
Wolff, S.M., J.H. Mulholland, andS.B. Ward. 1965. Quantitative aspects of the pyrogenic response of rabbits to endotoxin.J. Lab. Clin. Med. 65:268.
Lowry, O.H., N.J. Rosebrough, A.L. Farr, andR.J. Randall. 1951. Protein measurement with Folin phenol reagent.J. Biol. Chem. 193:265.
Kohlhage, H., W. Pollman, andR. Siegert. 1968. Studies on the differentiation of myxovirus induced interferon and endogenous pyrogen from rabbits.Life Sci. 7:627.
Hadley, W.K., J. O'Rourke, andE. Atkins. 1966. Purification and characterization of an endogenous pyrogen.Yale J. Biol. Med. 38:339.
Kozak, M.S., H.H. Hahn, W.J. Lennarz, andW.B. Wood, Jr. 1968. Purification and further chemical characterization of granulocytic pyrogen.J. Exp. Med. 127:341.
Gander, G.W., andF. Goodale. 1962. Chemical properties of leukocytic pyrogen. I. Partial purification of rabbit leukocytic pyrogen.Exp. Mol. Pathol. 1:471.
Rafter, G.W., S.F. Cheuk, D.W. Krause, andW.B. Wood, Jr. 1966. Studies on the pathogenesis of fever. XIV. Further observations on the chemistry of leukocytic pyrogen.J. Exp. Med. 123:433.
Murphy, P.A., P.J. Chesney, andW.B. Wood, Jr. 1971. Purification of an endogenous pyrogen, with an appendix on assay methods.In Ciba Foundation Symposium on Pyrogens and Fever, Churchill Livinsgtone, London. 59–69.
Murphy, P.A., J.P. Chesney, andW.B. Wood, Jr 1974. Further purification of rabbit leukocytic pyrogen.J. Lab. Clin. Med. 83:310.
Root, R.K., J.J. Nordlund, andS.M. Wolff. 1970. Factors affecting the quantitative production and assay of human leukocytic pyrogen.J. Lab. Clin. Med. 75:679.
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Dinarello, C.A., Wolff, S.M. Partial purification of human leukocytic pyrogen. Inflammation 2, 179–189 (1977). https://doi.org/10.1007/BF00917594
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DOI: https://doi.org/10.1007/BF00917594