Isolation and preliminary characterization of immunoinhibitory factors from dog erythrocytes and liver.

Crude dog liver extract (DLE) inhibits proliferation of dog and human lymphocytes stimulated by phytohemagglutinin and alloantigens. While purifying this activity from dog liver, we observed that dog liver inhibitory factor (DLIF) shared properties with hemoglobin. DLIF migrated with hemoglobin during DEAE cellulose chromatography, and DLIF had oligomeric (61,900) and subunit (17,900 and 15,700) apparent molecular weights (AMW) similar to concurrently analyzed Sigma canine hemoglobin (61,100, 16,700 and 14,900). After separate cross linking, the proteins comigrated on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) gels. We therefore isolated dog erythrocyte inhibitory factor (DEIF) from red blood cells to determine if DEIF was a hemoglobin derivative. DEIF, like DLIF, separated at an isoelectric point of 5.6. DEIF also had similar subunit AMW (17,600 and 15,300) by SDS PAGE, but DEIF had much lower lymphocyte inhibitory activity (LIA = 2.27) than DLIF (LIA = 100.00). We conclude that DLIF and DEIF are similar to each other and hemoglobin, but further studies are needed to determine the function and exact structure of DLIF and DEIF.

Variation in indirect cell-mediated lympholysis.

The ability of an allograft recipient to respond to donor mononuclear cells in an indirect cell-mediated lympholysis (ICML) assay is an in vitro correlate of allograft rejection, but the value of this correlation depends upon the assay's reliability. We had observed inconsistency in the cytotoxic response of normal human mononuclear cells (MNC) to the same allogeneic stimulator MNC when cytotoxicity was measured repeatedly on different occasions by micro-ICML. We, therefore, investigated the extent and reasons for this inconsistency. Method variation, determined by duplicate ICML of 18 stimulator: responder MNC, was not statistically significant. Variation in cytotoxicity over time was greater but still not statistically significant. The contribution to method variation of 51Cr release from 3 different sets of target cells, cultured and labeled in duplicate, was minimal (6.33%). We then asked if in vitro generation of effector MNC under laboratory conditions was a major cause of ICML variation. We tested this using a stable transplant's in vivo sensitized effector cells against donor MNC in a direct CML (DCML) and obtained consistent results. Finally, to gain an understanding of some of the factors which might influence the generation of in vitro cytotoxicity, we measured the frequencies of cell surface antigens (DR, TAC, transferrin, Leu 2 and 3) concomitantly with ICML on day 6 of culture. Statistical analysis of the results led us to conclude that the micro-ICML is reproducible. The magnitude of lysis depends upon activated target cells (TAC- and transferrin-positive) and an increase in the proportion of helper/inducer to cytotoxic/suppressor T-lymphocytes during effector cell generation.

Demonstration of a dog liver immunosuppressive factor active in vitro.

These studies define the presence of an immunosuppressive factor in dog liver that is active in vitro. A crude dog liver extract was prepared by saline extraction and ultracentrifugation. In vitro immunosuppressive activity of the extract was assayed by its influence on standard phytohemagglutinin mitogen stimulation assays (PHA), two-way mixed lymphocyte culture (MLC) reactions, and primed lymphocyte culture (PLC). In six experiments with four extracts, the mean percentage of inhibition (MPI) of dog lymphocytes in PHA mitogen stimulation assays was 102.0 +/- 3.6. In two human MLC experiments, the extract produced an MPI of 102.3 +/- 0.3. In one canine MLC the MPI was 102.5. In three PLC experiments the MPI was 92.7 +/- 8.0, indicating that the extract inhibited sensitization. Cytotoxicity of the extract was shown not to be the mechanism of proliferation inhibition by restimulation of cells washed free of extract, concurrent eosin viability studies, and documentation of normal base proliferation of cells after extract was washed from them. We concluded that there is a naturally occurring immunosuppressive agent in dog liver that is active in vitro as demonstrated by inhibition of PHA MLC, and PLC cellular proliferation assays. The activity is not attributable to cytotoxicity.