David Franks

Phagocytosis and killing of Trypanosoma dionisii by human neutrophils, eosinophils and monocytes.

The cell-mediated resistance of human leucocytes to Trypanosoma dionisii, a bat parasite related to T. cruzi, was investigated. Human peripheral blood neutrophils and monocytes were cytotoxic to T. dionisii as assessed by electron microscopy and by induction of 99mTc release from trypanosomes pre-labelled with [99mTc] pertechnetate. The enhancement of cytotoxicity by specific antiserum varied considerably from one individual to another. Neither blood lymphocytes nor blood eosinophils induced 99mTc release from T. dionisii. The trypanosomes were readily phagocytosed by neutrophils and monocytes even in the absence of added antiserum but the rate was enchanced when antiserum was present. Eosinophils also phagocytosed T. dionisii but only in the presence of antiserum. Investigation by electron microscopy revealed that T. dionisii is rapidly destroyed in the phagocytic vacuole of enutrophils and monocytes and by eosinophils. Phagocytosis, ultrastructural damage and induction of 99mTc release occurred more rapidly in neutrophils than in monocytes.

Killer cell (K) activity in human normal lymph node, regional tumour lymph node and inflammatory lymph node.

Human normal lymph nodes, irrespective of their anatomical site of origin, have a low K cell activity, which may not be detected except with the appropriate target cell and at high lymphocyte to target cell ratios (100:1). This very low killer cell activity is also found in all the homolateral axillary nodes of patients with clinical stage I and II carcinoma of the breast and in the regional draining nodes of a variety of solid tumours, whether small and localized or large and with extensive spread. In all cases proximity to the tumour and obious hyperplastic changes in the nodes have no modifying effect. This pattern of minimal reactivity is similarly found with tonsillar lymphocytes and with nodes draining inflammatory foci. The Fc and C3 receptors on surface membranes are dectected with ease, and pretreatment of lymphocytes by incubation, washing and enzymatic treatment fail to alter their reactivity, thus excluding effector cell inhibition by immune complexes. The killer cell activity of lymphocytes from the blood of breast tumor patients is similar to the activity of lymphocytes from healthy controls.

HORSE BLOOD GROUPS AND HEMOLYTIC DISEASE OF THE NEWBORN FOAL

The investigation of the red cell antigens of the horse a t Cambridge and Newmarket, England, was initiated by the discovery that hemolytic disease of the newborn foal is due to isoimmunization of mares during pregnancy (Coombs et al., 1948; Bruner et al., 1948). Eleven isoantisera, each with a different specificity, have been isolated from the sera of such mares thus far, and the characteristics of the in vitro reactions between the isoantigens and their respective isoantibodies have been studied (Franks, 1959). A summary of the serological characteristics of the antigen-antibody systems is given in TABLE 1. Only two of the isoantibodies, anti-3 and antid, produce agglutination of red cells suspended in saline; the reactions of the other isoantibodies can be detected by the antiglobulin test, by the use of enzymetreated cells, or by the use of serological tests depending upon complement fixation, such as hemolytic or conglutination tests. It is interesting that whilst both papain and ficin were able to make horse cells agglutinable by isoantibodies which have no effect on normal cells in saline, treatment of red cells with trypsin had no such effect. Inheritance of all the antigens thus far defined conforms with the hypothesis that each antigen is determined by a single gene, and that each gene is expressed in the heterozygote. A study of the frequencies of the 11 antigens shows that there are associations between antigens 1, 2, 5, 8, and 11, and between antigens 6 and 10 (TABLE 2). Investigation of the inheritance of these red-cell antigens shows that there must be close linkage between the 1, 2, 5, 8, and 11 genes, and no recombination has thus far been detected. The mating data involving the antigens determined by the genes a t this complex locus are summarized in TABLE 3. These genes appear to form a complex locus analogous to the human Rhesus complex or the cattle B locus. The 6 and 10 genes form another system, which was found independently by Podliachouk (1958) and called the A-F system by her. Estimates of the gene frequencies are given in TABLE 4.

Surface Characteristics of the Human K (Killer) Lymphocyte

The human non-allergized K (killer) lymphocyte has been characterized using selective depletion and isolation procedures based on surface markers. It was found to be a non-thymus-dependent, Fc-receptor-bearing cell. In an area which is controversial, our findings also indicate that the cell has immunoglobulin on its surface but lacks the receptor for the C3 component of complement (mouse). This would suggest it to be an (Fc + C3-) B lymphocyte.

Evasion of the oxidative microbicidal activity of human monocytes by trypomastigotes of Trypanosoma dionisii

Trypomastigotes of Trypanosoma dionisii, a stercorarian trypanosome from bats, are effectively killed by neutrophils from human peripheral blood but are less sensitive to the cytotoxic action of human monocytes. The mechanism of killing appears to involve peroxidase and hydrogen peroxide. Trypomastigotes are as effective as epimastigotes in inducing the formation of hydrogen peroxide by effector cells. They are, however, less sensitive than epimastigotes to the cytotoxic effect of peroxidase and hydrogen peroxide. They are therefore susceptible to the high concentrations of peroxidase found in the phagosome of the neutrophil, but resist the lower levels encountered in monocytes.

Inhibition of xenogeneic cell-mediated cytotoxicity in the rat by antiglobulin sera.

Differences can be demonstrated between the lysis of xenogeneic target cells by lymphocytes of injected rats, which is inhibited by antiglobulin serum, and the lysis of antibody-coated xenogeneic target cells by normal rat lymphoid cells, which is much less readily inhibited. This difference is demonstrated by the dilution of antiglobulin which is effective, and by the fact that inhibition of lysis produced by allergic lymphocytes can be shown if the lymphocytes are treated with antiglobulin and then washed. Differences can also be shown in the time course of inhibition, since to be effective antiglobulin must be present at the time of mixing normal lymphocytes and antibody-coated target cells, whereas addition of antiglobulin to allergized lymphocytes 4 h ater mixing with target cells still produces significant inhibition. These results are compatible with the hypothesis that allergic lymphocytes have immunoglobulin on their surfaces and kill by a different mechanism from the lymphoid cells which lyse antibody-coated target cells.

Effect of Prednisolone on Cell-Mediated Cytotoxicity in vitro

The effect of prednisolone on the kinetics of killing of Detroit-6 cells by rat lymphocytes has been studied utilizing the release of 51Cr as a measure of target cell death. The results show that in both killing by allergized lymphocytes, and killing of antibody coated target cells by normal lymphocytes, there is a short (less than 15 min) prednisolone-sensitive induction phase. Prednisolone has no effect on the reaction after this phase. The inhibition of induction by prednisolone appears to have two mechanisms. The first occurs rapidly following the addition of the drug to the cells, and must involve a primary action of prednisolone. Washing the cells free of prednisolone allows initiation, and killing can proceed almost normally, but if these washed cells are preincubated before mixing with target cells, then a secondary inhibition of induction develops progressively.

THE ROLE OF HYDROGEN PEROXIDE IN THE CYTOTOXICITY OF NEUTROPHILS TO TRYPANOSOMA DIONISII

Publisher Summary This chapter discusses the role of hydrogen peroxide in the cytotoxicity of neutrophils to Trypanosoma Dionisii. The chapter describes a study in which granulocytes and mononuclear cells were obtained from normal heparinized human peripheral blood by sedimentation with methyl cellulose and centrifugation through Ficoll–Hypaque gradients. The granulocyte fraction contained from 91 to 100% of granulocytes of which from 1 to 13% were eosinophils. The mononuclear fraction contained from 53 to 99% lymphocytes, the remainder being monocytes. Cytotoxic activity was measured from the ability of leucocytes to induce release of 99mTc from T. dionisii relabeled with 99mTc-pertechnetate in the presence of unlabeled sodium chromate. Leucocytes were mixed with T.dionisii at a ratio of 20 effector cells to one target and were centrifuged together into a pellet to induce close contact. After incubation for 3 h at 37°C, the amount of radioactive 99mTc released into the supernatant was measured. It was found that the granulocytes were more effective than mononuclear cells in inducing isotope release from T.dionisii. Cytotoxicity could be detected with granulocytes even at a ratio of one effector to one target.