Zoltan J. Lucas

Standardization of a sensitive and rapid assay for lymphotoxin.

Abstract Actinomycin-treated mouse L cells and human HeLa cells are sensitive indicators of lymphotoxin activity in supernatant fluids of mitogen-stimulated lymphocytes. Without actinomycin, various strains of these cells are 10–200 times less sensitive. The concentration of actinomycin used amplifies the toxic effect of LT but is not itself cytotoxic. Actinomycin-treated indicator cells permit detection of LT activity where toxicity is not often found, as in supernatants of mixed lymphocyte cultures and of cell-mediated cytotoxicity reactions. This assay makes available to any investigator a sensitive indicator of LT activity.

Immunological enhancement of renal allografts in the rat. 3. Role of the spleen.

SUMMARY F1 hybrid renal grafts survive for markedly prolonged periods in parental rat recipients treated by active or passive enhancement. Splenectomy of the renal recipient, when done 7 days before or up to 6 days after grafting, abolishes the immunological enhancement. Yet, splenectomy has no effect when performed 10 or more days later. Transferable enhancing activity is present as early as 2 days after a single i.v. immunization with allogeneic cells, preceding by 2–3 days both hemagglutinating and cytotoxic antibodies. Splenectomy prevents formation of enhancing activity but not hemagglutinating or cytotoxic antibodies.

Cytotoxic activity of lymphocytes: VIII. Lymphotoxin activity in cell-free extracts of activated lymphocytes

Abstract Cell-free extracts of human lymphocytes activated by PHA contain a cytotoxin that kills mouse L cells. Such cells elaborate two different kinds of toxins into the culture supernatant fluids, called α- and β-lymphotoxin (-LT), which differ in size, stability, and antigenicity. The amount of intracellular toxin is 1 to 24% of that found in supernatants of different tonsil donors. Equal amounts of intracellular toxin appear in both microsomal fraction (100,000g pellet) and soluble supernatant fractions of the cell-free extracts (CFE). The toxin can be solubilized from the membrane by digestion with papain or extraction with a nonionic detergent, but not by repeated sonication. The molecular weight of both the microsomal and soluble cellular cytotoxin is 45,000 ± 5000. The intracellular toxin differs from the extracellular toxins secreted by the same cells in two major characteristics: one, although its size approximates that of supernatant β-LT (and is smaller than the 76,000 M r α-LT), antibody-inhibiting α-LT but not β-LT inhibits both the microsomal and soluble CFE-LT. Two, the intracellular LT does not display the charge heterogeneity so characteristic of supernatant α-LT. Supernatant α-LT and CFE-LT are similar in their patterns of inactivation by heating to 80 °C and treatments with sodium dodecylsulfate (SDS), guanidine, proteases, and heavy metal ions, and are similarly unaffected by treatment with 8 M urea, N -ethylmaleimide, and sodium periodate. These results suggest that the single polypeptide intracellular LT is the precursor of the more complex secreted α-LT molecule.

Immunological enhancement of skin allografts in the rat. Role of vascular and lymphatic reconstitution.

Four skin graft models are created which vary in the rates of lymphatic and vascular reconstitution. Rat allografts (Brown Norway or (L × BN)F1 hybrid) transplanted to Lewis recipients by conventional (immediate lymphatic, delayed vascular), surgical anastomosis (immediate lymphatic, immediate vascular) or isolated anastomosis (delayed lymphatic, immediate vascular) techniques have similar mean survival times (7.8 days). Grafts placed on an isolated recipient pedicle (delayed lymphatic, delayed vascular) double the mean survival time to 15.6 days. Treatment with alloimmune serum, able to indefinitely prolong survival of similarly mismatched renal grafts, prolongs only isolated anastomosed grafts. In contrast, cyclophosphamide treatment prolongs survival of all groups. These results suggest, first, that both vascular and lymphatic routes of sensitization are equally effective and, second, that immunological enhancement requires either prompt vascular continuity or a persistent lack of lymphatic reconstitution.

Properties of an inhibitor of DNA synthesis in supernatants of activated lymphocytes

Abstract An 80,000 daltons non-cytotoxic inhibitor of DNA synthesis (IDS) of both mitogenactivated lymphocytes and HeLa cells was purified 28-fold from supernatants of 5 dayserum-free cultures of PHA-stimulated human lymphocytes. Purification was based on an assay measuring the inhibition of calf thymus DNA polymerase α utilizing denatured DNA in cell-free systems. Samples containing IDS activity inhibited a variety of DNA-template directed polymerases (calf thymus DNA polymerase α, DNA-dependent RNA polymerase from E. coli , and reverse transcriptase from avian myeloblastosis virus) suggesting that it acts on the DNA template rather than on the polymerase. In addition, single-stranded [ 3 H] OX174 DNA bound to IDS and the addition of IDS to native calf thymus DNA induced a hyperchromic shift in absorbance at 260 nm. IDP activity was distinguishable from contaminating lymphotoxin and RNase-H-like activities by heating to 70 °C for 15 min, a treatment destroying IDP only. The large size of IDS and its presumed intracellular site of action raise questions whether this lymphocyte factor functions to modulate DNA synthesis in the cell elaborating it or is specifically secreted to modulate replication of surrounding cells.

Microcytotoxicity assay for cell-mediated immunity enumerating residual target number by 86Rb

Lymphocyte-mediated lysis of target cells grown as monolayers in microtiter wells is readily quantitated by an assay measuring the 86Rb incorporated after attaining isotopic equilibrium. Lysis of fibroblasts by allogeneic lymphocytes sensitized by skin grafts and of tumor cells by syngeneic spleen cells sensitized by intraperitoneal tumor inoculation were readily detected. Weakly cytolytic lymphocyte populations can be assayed by increasing incubation times to 48 h or longer. A potential problem, 86Rb incorporation by lymphocytes sticking to residual target cells, was controlled by comparing 86Rb incorporation by targets incubated with non-immune lymphocytes. Results by 86Rb incorporation were identical to those determined by microscopic counting or 51Cr release. 86Rb incorporation assays should be considered as an alternate to 51Cr release techniques, especially in those experimental systems where the cytolytic potential of a lymphocyte population is so low that lysis can be detected only after long incubation times and/or when the spontaneous release of 51Cr is prohibitively high.

The Effect of Lymphatic Interruption and Immediate Vascularization on the Afferent Arc of Skin Graft Rejection

Allografted skin is more rapidly rejected than other organs such as kidney and heart, and its survival is less easily prolonged by immunosuppressive agents or by immunologic enhancement (1). Many explanations for this phenomenon have been given: 1) the rate of vascularization has been suggested to be important in view of the fact that the vascularization of skin grafts is delayed for several days, whereas whole organ grafts are immediately vascularized. Delayed vascularization may result in ischemic damage with greater vulnerability to immunologic attack (2); 2) skin has a relatively rich lymphatic drainage system which is reestablished post-transplantation. Barker and Billingham (3) have demonstrated that the lymphatics are the prime source of sensitization in skin. Similar findings could not be demonstrated in kidney grafts (4); 3) evidence is also accumulating that there is a quantitative and/or qualitative difference in the antigenicity of skin and other organs (5); 4) whole organs evoke more of a humoral than cellular reaction than skin, and may be autoenhancing.

Studies on the transformation of lymphocytes separated by suspension in dextran.

Abstract Phytohemagglutinin (PHA)-stimulated cells were suspended in 10% dextran to prevent cellular aggregation. Cells suspended in dextran did not respond to PHA by any of the five parameters tested: [ 3 H]leucine, [ 3 H]uridine, and [ 3 H]thymidine incorporation into acid-insoluble products, lymphotoxin synthesis, or morphologic transformation. Dextran itself was not cytotoxic to the lymphocytes. However, one major technical problem is attaining a proper dextran “control.” The difficulty in culturing lymphocytes in dextran without simultaneously causing some cell dispersion yielded dextran “control” values that showed less stimulation than cells without dextran. These results suggest that cell contact is necessary for PHA-induced transformation as judged by both DNA synthesis and lymphotoxin secretion. This technique now provides an experimental system for biochemically assessing the effects of antigens and mitogens without the attendant problems caused by cell clustering.

Role of the Spleen in Immunologic Enhancement of Kidney Grafts in Rats

Immunologic enhancement induced with alloimmune serum specifically suppresses renal allograft rejection in the rat (1, 2, 3). Several observations suggest that the recipient has an active role in enhancement. Alloantibody, rapidly fixed to tissues, disappears from the serum four hours after administration (4), and must be given at the time of grafting to insure graft survival (5). Multiple daily injections of suboptimal amounts are ineffective (5). Furthermore, enhancement of organ grafts, instead of preventing the immune response, modifies it so that the balance between graft rejection and prolonged survival is different from a primary graft response. Thus, hemagglutinating and cytotoxic antibodies appear after grafting with the same titer and kinetics as untreated grafts (3). Cell mediated immunity is similarly present (6).