Anti-MIF effects of a serum from a rabbit immunized with human lymphoid cell line migration inhibitory factor

WORKSHOP

ON

LYMPIIOCYTE

MEDIATORS

349

antigen-sensitive cells that release mediator (s) are most likely granulocytes ; the mediatorsensitive cells appear to include antigen-reactive T- and B-cells, but may comprise a broad cross section of cell types as part of the overall general stress response that includes the vascular and autonomic nervous system. .4nti-MIF Effects of a Serkruz from a Rabbit Iwnkrrkizrd with IIkkman Lynkphoid Cell Lirke Migration. Inlkibitory Factor. T. F. MCLEOD, B. L. BASKIN, S. K. MELTZ, C. F. SOROKIN, of Pediatrics, University of Miami School of Medicine, APED P. R. GLADE, Department Miami, Florida. The 50% (NHa)&OI-precipitable fraction of sera from a rabbit immunized with partially purified MIF obtained from a human B-lymphoid cell line (PGLC-33H) removes the MIF activity found in supernatants of this cell line. The Sephadex G-75 fraction containing supernatant MIF activity was applied to a preparative 8% acrylamide gel. The pre-albumin fractions from MIF supernatants and from control medium were lyophilized. suspended in Freund’s adjuvant, and injected at monthly intervals into rabbits. The sera collected following the fourth and fifth injections showed no direct effect on the migration of PGLC-33H target cells, nor did they directly block the migration inhibition induced by PGLC-33H supernatants. By Ouchterlony technique, the serum and 50% (NH,)&Ot-precipitable fraction from the immune rabbit only demonstrated a line of identity with fetal bovine serum and bovine serum albumin and a line of partial identity with human serum. Assay of Sephadex G-75 and polyacrylamide fractions of supernatant MIF previously incubated with (NH2)rS04 precipitates of sera showed that: (1) incubation with immune rabbit precipitates removed the MIF activity observed in fractions 5-6; (2) control rabbit precipitates did not alter the detection of MIF; (3) incubation with precipitates from sera taken before immunization had no effect on MIF activity. ~‘kruificatiokk SMITH

Branch,

and Charactcuizatiokk

of Biosynthctical/y

AND B. W. PAPERMASTER,

Galveston,

Division

Labeled of Biochemistry,

hfokrse Lynzphotoxin. University of Texas

hf. E. Medical

Texas.

Concentrated supernatant culture medium from the L1210 lymphoma has been shown to contain a cytotoxic factor active against several target cell lines. Cytotoxicity was measured in assays monitoring uptake of vital stains, inhibition of uptake of [‘Hlthymidine, release of cptosol lactate dehydrogenase, and inhibition of in VWO tumor growth. Using L1210 cells grown in media containing high specific activity [“HIamino acids, endogenously labeled fractions have been produced which allow characterization and purification of the biosynthetically produced cytotoxic factors. Purification procedures involved gel filtration, preparative agarose gel electrophoresis, and immunoadsorbents. Purification is hindered by the apparent association of both biological activity and endogenous ‘H label to bovine albumin and another large molecular weight carrier molecule in the culture media. Reversible dissociation of the cytotoxic factor from these carrier molecules is shown and this property is utilized for purification in affinity adaptive chromatography. Molecular weight determinations as done by SDS polyacrylamide gel electrophoresis and gel filtration in 6 M guanidine indicate a molecular weight range of 20,000-40,000 for the purified dissociated cytotoxin factor. (Supported in part by DHEW lPO1 C-4 16964-01 and the James W. McLaughlin Fellowship Fund.) Pcvitorkral

Fluid Factor H. I?. DVORAK, Massachusetts. AND

That Inhibits Cell Adhevem?. M. J. GAI.T..%GHER, M. E. HAMMOND, Department of Pathology, Massachusetts General Hospital, Boston,

Washings of the normal guinea pig peritoneum contain a factor(s) that inhibits the sticking of macrophages and of other cells including line 1 tumor cells to glass and to the isolated mesentery. Cell sticking is expressed as the numher of adherent cells in 10 high power fields in a chamhcr mounted on a microscope slide. The factor. PAIF (peritoneal adherence inhibition factor), is also present in tumor ascites and, in increased amounts, in oil-induced peritoneal exudates; activity is largely lost after clotting. A similar activity is present in plasma and in much smaller amounts in serum. PAIF is inactivated by pronase, but not by chondroitinase; sediments as a protein on Cccl2 density gradients; and is soluble at pH 3.3, condi-