Cytotoxicity Of Leukocytes Research Articles

Antibody-dependent cellular cytotoxicity of leukemic cells from the patients with acute myeloid leukemia to human erythrocytes coated with anti-D antiserum.

We examined the antibody-dependent cellular cytotoxicity (ADCC) of normal leukocytes from healthy volunteers, immature neutrophils from 4 patients with chronic myelocytic leukemia (CML) and leukemic cells from 22 patients with acute myeloid leukemia, employing human erythrocyte targets. Ability to mediate ADCC was demonstrated only in monocytes among normal leukocytes and in leukemic cells from all the 6 patients with acute myelomonocytic or monocytic leukemia (AMMoL, AMoL). CML immature neutrophils and leukemic cells from 14 patients with acute myeloblastic or promyeloblastic leukemia displayed very low levels of ADCC. In the remaining 2 patients with acute myeloblastic leukemia, the ADCC levels were high. We described the usefulness of this ADCC assay for distinguishing AMMoL or AMoL from other types of acute myeloid leukemia.

Natural and antibody-dependent cellular cytotoxicity of polymorphonuclear leukocytes.

Cytotoxic activity of polymorphonuclear leukocytes (PMN) in the peripheral blood of patients with various diseases was demonstrated to K562 cells (natural cytotoxicity, NC) and the antibody-coated P815 cells (antibody-dependent cellular cytotoxicity, ADCC), using a 51Cr-release method. The NC values of normal PMN were lower than those of normal lymphocytes with mean values of 5.0% and 30%, respectively. The NC values of patients' PMN were also lower in malignancy, chronic hepatitis and connective tissue diseases. The ADCC values of normal PMN were moderately high with a mean value of 16.0%, which was almost a half of normal lymphocytes. Higher ADCC values of PMN were found in patients with chronic hepatitis, SLE and Behcet's disease, and in these cases the ADCC values of their lymphocytes were extremely low. The supernatants of PMN mix-cultured with unlabeled K562 or the antibody-coated P815 cells were fairly cytotoxic to both cells, though the similar supernatants of lymphocytes were cytotoxic only to K562 cells, but not to the antibody-coated P815 cells.

Inhibition of cellular cytotoxicity of leukocytes for herpes simplex virus-infected cells in vitro and in vivo by intralipid.

The effect of intralipid, a lipid emulsion used in total parenteral nutrition, on cellular cytotoxicity for herpes simplex virus (HSV)-infected cells was analyzed. In vitro, intralipid inhibited antibody-dependent cellular cytotoxicity (ADCC) of lymphocytes, monocytes-macrophages, and polymorphonuclear leukocytes and natural killer cytotoxicity of lymphocytes for radiolabeled HSV-infected liver cells. This was due to an effect on the leukocytes, rather than on the target cells. Intralipid did not affect leukocyte viability but inhibited the expression of leukocyte Fc receptors necessary for cytotoxicity. In vivo, intralipid inhibited murine ADCC and completely nullified the protection against lethal infection with HSV in neonatal mice afforded by the administration of human leukocytes and antibody. These data suggest that high levels of circulating intralipid may interfere with antiviral immunity in humans and predispose hosts who are already compromised to severe viral infections.

Elimination diets in the treatment of secretory otitis media

The significance of elimination diets in the treatment of secretory otitis media (SOM) and the usefulness of the cytotoxic leucocyte test (CLT) in the planning of elimination diets were investigated. Sixty-seven children with SOM were tested with CLT and treated with or without elimination diets in addition to the established SOM-treatment. After two years of observation in patients with multiple CLT-reactions (50) there was an almost significant difference ( P < 0.06) in the cure rates and a significant difference ( P < 0.02) in the percentages of therapeutic failures between those treated with (32) and those treated without (18) elimination diets. No marked difference was found between patients, who did not have multiple CLT-reactions (17) and were treated without diets and patients who were treated with diets for multiple CLT-reactions (32). It was concluded that elimination diets are needed in the treatment of SOM for those patients who have multiple reactions in the CLT and that the CLT is useful in the planning of these elimination diets.

Pulmonary Complications during Granulocyte Transfusions: Incidence and Clinical Features

The use of granulocyte transfusions in profoundly neutropenic patients has increased markedly in recent years. Whenever a pulmonary infiltrate develops during the course of these transfusions, the question arises as to what role the transfusions are playing and whether the transfusions should be discontinued to prevent pulmonary deterioration. We have analyzed our recent experience of 593 granulocyte transfusions in 93 patients. 18 patients (19%) developed respiratory compromise or pulmonary infiltrates at some time during the course of granulocyte transfusion. 6 of the 18 cases were reactions to the granulocytes while the remainder were due to fluid overload or other causes. The risk of pulmonary complications did not correlate with the development of cytotoxic leukocyte antibodies, length of transfusion, or concomitant use of Amphotericin. They appeared to be more common in patients with active sepsis. Acute life-threatening pulmonary reactions were rare. Patients receiving granulocyte transfusions should be monitored carefully for pulmonary infiltrates, but other cases should be sought before the transfusions are discontinued.

Separation of cytotoxic leukocyte populations of human peripheral blood and colostrum on PVP-silica (Percoll®) density gradients

PVP-silica (Percoll) did not inhibit antibody-dependent cellular cytotoxicity in an 18 h 51Cr-release assay. By the use of a simple discontinuous density gradient centrifugation technique, leukocytes from peripheral blood and colostrum were separated into morphologic and cytotoxic functional populations. The major cytotoxic effector cells in colostrum are lipid-free macrophages and probably a small lymphocyte subpopulation.

Genetically restricted cell-mediated cytotoxicity in cattle immune to Theileria parva.

The protozoan parasite Theileria parva, which is transmitted by the tick Rhipicephalus appendiculatus, produces an acute fatal infection in the lymphoid system of susceptible cattle1. This disease is a serious constraint to livestock improvement and production in large areas of East Africa1. The parasite invades host lymphocytes, inducing rapid proliferation followed by widespread lymphocytolysis2. Cattle which recover from theileriosis (East Coast fever) spontaneously, or which are immunized by infection and treatment with tetracycline3, are resistant to reinfection with the same isolate of T. parva for at least 3 yr4. Immunity against infection with the parasite cannot be ascribed to the production of specific antibodies5, but can be transferred adoptively between twins with thoracic duct leukocytes from the immunized partner6. These observations suggest that protective immunity is associated with cell-mediated mechanisms. We have now examined the capacity of leukocytes from immune cattle to lyse parasitized lymphoblastoid and non-parasitized tumour cell lines either directly or after stimulation in an autologous mixed lymphocyte reaction (MLR). In contrast to the nonspecific lytic activity of leukocytes from immune cattle reported by Pearson et al.7, we describe the sequential appearance in the lymph and blood of immune cattle, of cytotoxic leukocytes with activity restricted to target cells carrying the autologous genotype. These observations suggest that a major component of protective immunity to T. parva is mediated by cytotoxic cells which lyse parasitized cells in a genetically restricted fashion. We have also found that during a primary infection with T. parva cytotoxicity was manifested against allogeneic parasitized cells and xenogeneic uninfected target cells, but not against autologous infected cells. The features of cell-mediated immunity to T. parva during primary infection and immunization are discussed more fully elsewhere8.

Secretory Otitis Media and Allergy

This study is an investigation on whether allergy, and especially food allergy, is more common in patients with secretory otitis media (SOM) than in others. At the same time, the effectiveness of the cytotoxic leucocyte test (CLT) as an aid in the diagnosis of food allergy was also studied. The material comprised 90 patients: 69 suffering from secretory otitis media and 21 without the disease as controls. Patients were studied using allergy history, ear status, tests for nasal and blood eosinophilic cells, total IgE, skin tests for 20 allergens. RAST for milk and wheat, immunoglobulin A, G and M, precipitating antibodies for milk and gluten. Mantoux test, and the CLT for 23 allergens. Allergy was found in 20% of the SOM patients and 10% of the control patients; the difference was not statistically significant. The best methods for verifying allergy were history, skin tests, and tests for nasal eosinophils cells. Laboratory tests could not verify objectively that food allergy was more common among SOM patients than controls, although 16% of the SOM patients compared with none of the controls had a history of food allergy. Of the tests used, the CLT correlated best with a history of allergy, but the results of the different tests were not in accordance wtih each other.

Human colostral cytotoxicity. II. Relative defects in colostral leukocyte cytotoxicity and inhibition of peripheral blood leukocyte cytotoxicity by colostrum.

Colostral cells have been previously shown to mediate antibody-dependent cellular cytotoxicity (ADCC) to herpes to simplex virus-infected cells. A comparison of colostral cells to matched peripheral blood mononuclear cells has revealed significantly lower ADCC activity, higher antibody requirements, and slower kinetics of colostral cell ADCC against infected cells. Colostral cells failed to mediate natural killer cytotoxicity. Lymphocytes, monocyte macrophages, and polymorphonuclear leukocytes isolated from peripheral blood and incubated with colostrum from virus-immune or nonimmune women markedly inhibited ADCC. Inhibitory activity was found in lipid and aqueous fractions and was due to an effect on leukocytes, not target cells. A partial explanation was inhibition of expression of leukocyte Fc receptors, a prerequisite for ADCC, by acellular colostrum. These results raise questions concerning the potential antiviral activity of colostral cells in vitro.

Effect of antineoplastic drugs on human leukocyte-mediated cytotoxicity against herpes simplex virus infected cells.

We evaluated the effect of five antineoplastic drugs on the ability of human leukocytes to destroy herpes simplex virus (HSV) infected target cells in the presence of antibody (antibody-dependent cellular cytotoxicity) and in its absence (natural killer cytotoxicity). Leukocytes from healthy volunteers were separated into macrophages, polymorphonuclear leukocytes, and lymphocytes. Adriamycin, cyclophosphamide, prednisone, procarbazine, and vincristine, at various concentrations and incubation periods, were tested for their effects on the natural killer and antibody dependent cellular cytotoxicity of macrophages, lymphocytes, and polymorphonuclear lymphocytes in a 51Cr release microcytotoxicity assay against HSV-infected cells. All drugs at therapeutic concentrations inhibited natural killer and antibody dependent cellular cytotoxicity; an exception was cyclophosphamide, which did not inhibit the natural killer cytoxicity of lymphocytes. The antibody dependent cellular cytotoxicity of macrophages and polymorphonuclear leukocytes appeared to be more than that of lymphocytes. The results of short incubation (2 hours) of the drug with either effector cells or target cells, followed by drug removal, suggests that the drug effect occurred early and predominantly at the effector cell level. Antineoplastic drugs had an inhibitory effect on natural killer and antibody dependent cellular cytotoxicity against HSV-infected cells. This inhibitory action may partially explain the increased susceptibility of patients receiving chemotherapy to developing viral infections.

Cytotoxic leukocyte reaction.

Blood samples from 49 subjects were submitted to cytotoxic leukocyte tests, which were read double-blind by two laboratory technicians. The results were identical in 78% of the tests and the statistical error of the method was 0.46. In a group of 32 subjects, the cytotoxic test was repeated on 3 consecutive days. Deviations in the results occurred in 18--20% of the tests, the statistical errors varying between 0.38 and 0.43. In a series of serum samples (75 subjects) with positive leukocyte reactions to food antigens, various in vitro procedures were carried out in connection with the cytotoxic test. The following observations were made: In 42 of 58 tests, a positive leukocyte reaction was transferred with the reactive serum to cells from non-reactive subjects. After heating of the reactive serum (56 degrees C, 2 h) and washing of the cells, reactivity was no longer observable in 44 out of 52 tests. Treatment with EDTA consistently blocked expected cytotoxic reactions in 36 tests. DSCG inhibited expected reactions in 45%, antihistamine in 93%, and cortisone in 20% of the tests.

Interferon and cell-mediated immunity

The thesis is introduced that the phenomena of cell-mediated immunity proceed with the necessary involvement of interferon. In view of this thesis various examples of cell-mediated cytotoxicity both antibody-dependent, and antibody-independent are analyzed. The role of interferon in transplantation, allergic diseases and the treatment of cancer is also discussed. Based on this analysis it is concluded that interferon provides for the cytotoxicity of immune and non-immune leukocytes. It is suggested that interferon is responsible for the cytotoxic effect in itself, since it facilitates lethal hybridization. Recognition of the important role of interferon in the mechanism of cell-mediated immunity leads to some practical conclusions: a) The selective inhibition of the activity of type II interferon may be a sufficient measure in cases where now total immunosuppression is applied. b) Periodic treatment of healthy population by lymphokine stimulants may be a method of prevention of cancer. c) Transplantation, allergic diseases, immunotherapy of cancer should be considered in terms of the role of interferon in these processes.

Leukocyte cytotoxicity in a persistent virus infection: presence of direct cytotoxicity but absence of antibody-dependent cellular cytotoxicity in horses infected with equine infectious anemia virus

Antibody-dependent cellular cytotoxicity and direct cytotoxicity assays were performed with equine infectious anemia virus-infected target cells, equine leukocytes, and equine anti-equine infectious anemia virus antibody to determine whether these mechanisms play a role in controlling viral replication in equine infectious anemia. Direct cytotoxicity was observed by using peripheral blood mononuclear cells from 7 of 10 infected horses. Antibody-dependent cellular cytotoxicity was not observed. The antibody-dependent cellular cytotoxicity reaction in horses was then studied by using sheep erythrocytes and trinitrophenylated sheep erythrocytes as target cells. Lysis of these target cells was mediated by neutrophils, monocytes, and lymphocytes. The reaction was activated by antibody of the immunoglobulin G class but not by immunoglobulin G(T). Furthermore, immunoglobulin G(T) efficiently inhibited immunoglobulin G in this function.

The Inhibition of Polymorphonuclear Leukocyte Cytotoxicity by Dapsone

The effect of the sulfone compound 4,4'-diaminodiphenyl sulfone (dapsone) on normal human polymorphonuclear leukocytes (PMNL) has been investigated in vitro. The drug has a dramatically beneficial effect in dermatitis herpetiformis in which the PMNL and immune complexes has been stressed to be of importance for the development of the skin lesions. Pruritus disappears and the inflammatory eruptions clear within a few days of starting therapy. The effect of dapsone has been evaluated on the different stages of phagocytosis. Using dapsone concentrations (1-30 mug/ml) comparable with those found after therapeutic doses, we have found that the drug interferes primarily with the myeloperoxidase (MPO)-H(2)O(2)-halide-mediated cytotoxic system in the PMNL. No effect was observed on random locomotion, chemotaxis, phagocytic ingestion, oxidative metabolism, or the release of lysosomal enzymes. Kinetic studies in a cell-free system with purified MPO revealed a competitive type of inhibition using varying concentrations of NaI. Furthermore, the inhibition resulted in reduced candidicidal activity during phagocytosis of Candida albicans, and reduced cytotoxicity to adjacent mammalian cells measured as the (51)Cr release from virus-induced lymphoma cells. Because the MPO-H(2)O(2)-halide system not only fulfills the antimicrobial activity but is suggested to be a modulator of the inflammatory reaction as well, the action of dapsone in dermatitis herpetiformis may in part be explained by its effect on this system.

The partial isolation and function of salivary factors which interact with the complement system: a possible role in mucosal immunity.

Penetration of oral bacterial substances, coated with salivary constituents, into damaged mucosa and subsequent contact with serum complement components is an example of the potential for saliva-complement reactions. These interactions may affect not only the production of the potent complement-generated anaphylatoxins and chemotactic factors, C3a and C5a, but also other immunological responses which depend to varying extents on complement activation. These include stimulation of the differentiation of B lymphocytes to become antibody-producing plasma cells, B-cell-mediated cytotoxicity, and stimulation of macrophages and polymorphonuclear leukocytes to release lysosomal proteolytic enzymes. Stimulation of these cells is thought to occur as a result of the attachment of complement-coated antigens to specific C3b and C5b receptors on B lymphocytes, macrophages, and polymorphonuclear leukocytes. Furthermore, saliva-coated oral antigens may contact the functional complement components in the crevicular fluid (1) prior to tissue penetration.

Cytotoxicity of circulating leukocytes in cytomegalovirus mononucleosis

Leukocyte cytotoxicity was studied in a patient with cytomegalovirus (CMV) mononucleosis. Circulating leukocytes demonstrated marked cytotoxicity for Raji cells, an Epstein-Barr virus (EBV)-containing cell line which is free of detectable amounts of CMV DNA. Cytotoxicity correlated directly with the percentage of atypical lymphocytes. Similar cytotoxic responses occur in patients with EBV mononucleosis and may represent a specific response to a common cellular antigen. Alternatively we suggest that there is activation of a common cytotoxic effector cell evidenced by the atypical lymphocytosis. The similarity of monoucleosis caused by EBV or CMV may then result from broad tissue-damaging effects of such activated cells.

Inhibition of lysosomal enzyme release from rat leukocytes by auranofin. A new chrysotherapeutic agent.

Auranofin (SK&F D-39162), a new antiarthritic gold compound reported to be orally effective in animal (adjuvant rat) and human (rheumatoid) arthritic conditions, is a potent in vitro inhibitor of the release of lysosomal enzymes from phagocytizing rat leukocytes. Auranofin, at micromolar concentrations (1-10 microM), produced a dose-dependent reduction in extracellular levels of lysosomal enzyme markers (beta-glucuronidase and lysozyme) which are selectively released from rat leukocytes during phagocytosis of zymosan particles. The reduction in extracellular levels of lysosomal enzymes appears to be caused by inhibition of their selective cellular release, since effective concentrations of auranofin did not produce leukocyte cytotoxicity or inhibition of cell-free lysosomal enzyme activity. Morphologic and biochemical evidence indicated that auranofin also interferes with phagocytosis of zymosan particles. The potent in vitro activity of auranofin appears to result from its unique gold complex, since neither structurally related nongold compounds nor clinically used gold compounds (gold sodium thiomalate and gold thioglucose) were potent inhibitors of lysosomal enzyme release. The results of this investigation suggest that the antiarthritic activity of auranofin may be caused at least in part, by inhibition of lysosomal enzyme release and/or cellular processing of antigens.

The cytotoxicity of leukocytes and lymphocytes from patients with rheumatoid arthritis for synovial cells.

Unseparated peripheral blood leukocytes obtained from patients with rheumatoid arthritis (RA) were cytotoxic for synovial cells. The cytotoxic reactions produced by RA leukocytes were more frequent and of greater magnitude than cytotoxicity induced by leukocytes from normal persons and patients with other diseases, primarily connective tissue diseases. Furthermore, the cytotoxic activity of RA leukocytes was greater for RA synovial cells than for nonrheumatoid synovial cells, in contrast to the cytotoxicity of other leukocytes, which did not discriminate between synovial cells according to their origin. Tests with purified lymphocytes showed that the cytotoxicity of unseparated leukocytes directed against RA synovial cells was due to lymphocyte cytotoxicity. These data are consistent with the possibility that sensitized lymphocytes from patients with RA recognize a distinctive antigen present on rheumatoid synovial cells.

Effect of Six Weekly Transfusions on Canine Marrow Grafts: Tests for Sensitization and Abrogation of Sensitization by Procarbazine and Antithymocyte Serum

We have previously shown that blood transfusions can immunize a dog and lead to rejection of a subsequent marrow graft despite lethal total body irradiation (TBI). Sensitization to histocompatibility antigens induced by two prior transfusions of whole blood could be overcome by a regimen of procarbazine and anti-thymocyte serum (ATS) preceding TBI. The current study investigated a) whether this regimen could abrogate sensitization induced by six weekly transfusions given from days --50 to --15 preceding a marrow graft, and b) whether platelet survival studies and two in vitro tests of immunity could predict marrow graft rejection. All donor-recipient pairs were histoincompatible, unrelated, and of different breed. Twenty-two recipients received platelet concentrate transfusions and eight received whole blood transfusions. Recipients were given 1200 R TBI and a graft of marrow and peripheral blood leukocytes from the transfusion donor on day 0. Three of 15 recipients (20%) given procarbazine, 12.5 mg/kg i.v. on days --8, --6, and --4, and ATS, 0.6 ml/kg subcutaneously on days --7, --5 and --3, Rejected their grafts, whereas 11 of 15 dogs (73%) not given procarbazine and ATS rejected their grafts (p less than 0.01). Serum lymphocytotoxic antibodies, peripheral leukocyte migration inhibition, and in vivo donor platelet recovery and survival were studied in those recipients receiving six weekly transfusions and in 18 other recipients receiving a single donor transfusion 3 months before marrow grafting. No significant correlation was found among these in vitro and in vivo tests of sensitization. Sensitization to marrow grafts was not reliably detected by the presence of cytotoxic antibodies or leukocyte migration inhibition. Platelet survival, however, was positively correlated with the results of marrow grafting in 12 of 15 (80%) evaluable recipients (p approximately 0.15).

Differing time courses of spleen leukocyte MIF synthesis and cytotoxicity during rejection of a murine lymphoma allograft

In vitro spleen leukocyte MIF synthesis and direct cytotoxicity were studied during growth and rejection of the EL-4 murine lymphoma in allogeneic tumor-bearing BALB/c mice to compare the time courses of these events. Rejection of EL-4 tumors occurred between 6 and 8 flays after intraperitoneal inoculation. Spleen leukocyte cytotoxicity measured by isotope release from chromium-51 labeled EL-4 target cells was first detected on day 6 and was maximal between days 11 and 18. In contrast, spleen leukocyte MIF synthesis stimulated by intact EL-4 cells was sometimes observed on day 11 and was maximal between 18 and 25 days after tumor challenge. These results show that maximal spleen cytotoxicity and MIF synthesis occur after completion of, rather than during ip tumor rejection and, in addition, that these two in vitro lymphocyte responses follow independent, significantly different time courses ( P < 0.05). This asynchrony of MIF synthesis and cytotoxicity suggests that these in vitro correlates are mediated by distinct lymphocyte subpopulations.