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Phagocytic Activity of the Leukemic Cell and Its Response to the Phagocytosis-Stimulating Tetrapeptide, Tuftsin1

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Phagocytic Activity of the Leukemic Cell and Its Response to the Phagocytosis-Stimulating Tetrapeptide, Tuftsin1 [CANCER RESEARCH 33, 1230-1234, June 1973] Phagocytic Activity of the Leukemic Cell and Its Response to the Phagocytosis-stimulating Tetrapeptide, Tuftsin1 Andreas Constantopoulos, Vilas Likhite, William H. Crosby, and Victor A. Najjar Division of Protein Chemistry [A. C., V. A. N.J, Department of Molecular Biology and Microbiology, Tufts University School of Medicine, and The Department of Pediatrics [V. A. N.] and Medicine [V. L., W. H. CJ, New England Medical Center, Boston, Massachusetts 02111 SUMMARY important role of certain specific cytophilic 7-globulins in stimulating phagocytes. A leukophilic fraction of -/-globulin, The phagocytic activity of three types of leukemic cells was termed leukokinin (5, 6) coats the PMN3 cell and enables it to studied for Staphylococcus aureus in physiological Krebs- exhibit a high level of phagocytosis. A study of the mechanism Ringer phosphate glucose medium alone, and after stimulation of leukokinin action on the PMN cell revealed that its with autologous complement-inactivated serum and with biological activity resides fully in a simple peptide which forms phagocytosis-stimulating tetrapeptide, "tuftsin." The level of an integral part of the leukokinin molecule. This peptide, tuftsin activity in the sera of patients was also evaluated. which we have christened "tuftsin," (7, 9) has been isolated, Fifteen patients were included in this study: six with its structure determined, and the compound syntheszied (9). It myelofibrosis, seven with acute granulocytic leukemia, and is composed of 4 amino acid residues with the sequence of two with myelomonocytic leukemia. In all six cases of threonyllysylprolylarginine. It is active in hormone-like quanti myelofibrosis, the polymorphonuclear cells showed a normal ties and was found to be deficient in certain patients with level of basal phagocytosis in Krebs-Ringer phosphate glucose repeated infections, in those with "tuftsin deficiency syn medium. Five of seven cases of acute granulocytic leukemia drome" (3), and in splenectomized subjects (7). showed normal or near-normal values also, while two had The phagocytic activity of PMN leukocytes from patients significantly diminished levels. All thirteen of these patients with acute granulocytic leukemia has been investigated failed to show stimulation with saturating amounts of repeatedly with variable results (1,10,13, 14,16, 17). Studies autologous serum or tuftsin. This is distinctly abnormal, since of patients with myelofibrosis have not been reported. Most normal blood granulocytes of humans, dogs, and rabbits are studies of PMN granulocytes were performed in the presence considerably stimulated. The two cases of myelomonocytic of autologous or normal serum, circumstances in which a leukemia available for study showed a higher than normal deficiency of certain humoral factors such as leukokinin or basal phagocytic activity in Krebs-Ringer phosphate glucose tuftsin could have been missed. It has not been possible to medium and responded normally to serum and tuftsin determine whether the phagocytic deficiency was inherent in stimulation. the granulocyte itself or involved serum factors, or both. Since we now differentiate between these possibilities, a study was undertaken of leukocytes from patients with acute granulo INTRODUCTION cytic leukemia and with myelofibrosis. First, we examined the responsiveness of the PMN cells of these patients to phagocytic It is generally recognized that infection is a major cause of stimulation by complement-inactivated autologous serum and death in patients with WBC dyscrasias (4, 12, 15, 18). The synthetic tuftsin. Second, we investigated tuftsin levels in basic abnormalities of the disease are often worsened by serum during the active stage of the disease. chemotherapeutic insults which predispose the patient to a variety of repeated and often severe infections. Drug therapy has considerably prolonged the life-span of many patients, but MATERIALS AND METHODS optimal management continues to depend on the joint mobilization of antileukemic and antimicrobial therapy. Seven patients with acute granulocytic leukemia, 2 with Patients with advanced myelofibrosis become susceptible to acute myelomonocytic leukemia, and 6 with myelofibrosis infection as do patients in late stages of granulocytic .leukemia were studied (Table 1). The diagnosis was based on clinical (11). findings, peripheral blood films, and bone marrow morphol During the past few years we have investigated the ogy, along with cytochemical identification of granulocytes and monocytes (19). In all patients with myelofibrosis, bone 'Supported by Grant AI-09116, NIH, USPHS.and Grant GB-31535 marrow biopsies were performed. Twelve healthy individuals X, National Science Foundation. of both sexes, 24 to 68 years old, were used as controls. 'American Cancer Society Professor of Molecular Biology (MA Usually, 15 ml of heparinized blood and 5 ml of clotted blood Division), to whom reprint requests should be addressed, at Division of Protein Chemistry, Tufts University School of Medicine, 136 Harrison Avenue, Boston, Mass. 02111. 'The abbreviations used are: PMN, polymorphonuclear; KRPG, Received August 14, 1972; accepted March 5, 1973. Krebs-Ringer phosphate glucose. 1230 CANCER RESEARCH VOL. 33 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1973 American Association for Cancer Research. Phagocytosis by Leukemia Cells and Tuftsin Effect Table 1 Clinical description of patients used in phagocytosis studies This table depicts 15 patients with blood dyscrasias. The total number of white blood cells, along with the relative percentage of the mature and immature forms, are also included. The state of the disease, whether in remission or relapse, is given in the last column. Peripheral blood picture Total no. of phagocytes/ WBC/ eu mm PatientR. (yr)435376482037655373726465765071DiagnosisAcuteMNÛ)90,70080,00010,9004,1003,4805,20083,6002,70043,0008,40083,6007,2003,50026,0002,4009,97716,0002183,0751,46131245,9801,89017,2005,37620,9006,0482,87015,6001,680PMN(%)920272406516738512268825051MNeu mm (PMN + (%)2NoneNone32None43213316None1019L<%)25NoneNone2558664302982816182430Blast(%)648098NoneNone2841None8None24NoneNone16NoneP(%)NoneNoneNoneNoneNoneNoneNoneNone14413NoneNoneNoneNoneM<%)NoneNoneNoneNoneNoneNoneNoneNone9410NoneNoneNoneNoneRemarksRelapseRelapseRelapseRemissionRemissionRelapseRelapseRemissionRelapseRelapseRelapseRemissionRemissionRelapseRemission A.M. C.E. D.A. granu-locytic F.J. leu H.W. kemiaMyelofibrosisAcute K.H. M.G. B.W.U.V.M.L. P.W. P.F. S.A. P.L. myelo-monocyticleukemiaTotal S.SexMFFMMMFMMFFMMFMAge 1The abbreviations used are: MN, monocyte; L, lymphocyte; P, promyelocyte; M, myelocyte. min in silicon-coated glass-stoppered tubes at 37°with continu were drawn from each patient and from control subjects. The serum was inactivated at 56° for 30 min. The synthetic ous shaking by tumbling at 8 cycles/min in a circular rotor. The tetrapeptide (9) was used throughout for phagocytic stimula components of the reaction mixture were added rapidly within tion. All glassware used in the procedure detailed below was 1 min and consisted of: (a) 0.1 ml of white cells; (b) 0.1 ml freshly siliconized. PMN leukocytes were obtained by sedimen KRPG medium or 0.1 ml (6 nmoles) tuftsin in the same tation in 0.15% dextran or by centrifugaron of heparinized medium; 0.1 ml autologous, inactivated deopsonized serum; or blood at 150 X g for 10 min and harvesting the buffy coat at 0.1 ml representing 2.0 mg of 7-globulin in KRPG medium, as the top of sedimented cells. The white cells were washed 3 needed in the test; (c) 0.1 ml bacteria to start the reaction. times with 10 volumes of KRPG medium, containing 2 mg of A loopful of the mixture was removed at the end of the glucose per ml, pH 7.4. This was done in order to remove the reaction and mixed with another loopful of bovine serum leukophUic 7-globulin coat. The cells were then taken up in albumin (60 mg/ml), smeared, air dried, and stained with the same medium containing 3 mg of bovine serum albumin Wright's solution. In all cases, 200 to 400 PMN cells were per ml (3, 7, 9). The final cell count was 2 X IO7 cells/ml and counted independently by 2 observers. The phagocytic index included all myelocytic cells at any stage of maturation. is recorded as the number of cells, belonging to the PMN Coagulase-positive Staphylococcus aureus was used as the series, containing one or more S. aureus per 100 cells observed. target particle. The bacteria were cultured for IE hr in Phagocytic stimulation by serum or tuftsin is the index heart-broth infusion. An aliquot was sedimented and thor observed in the presence of the KRPG medium with added oughly opsonized for 30 min at 37°with 1 ml of normal serum or tuftsin, minus the reagent control, i.e., the basal inactivated human serum of high opsonin content. After phagocytic index obtained with KRPG medium with no having been washed 3 times with KRPG medium, the bacteria additions. In this system, maximal values of phagocytic index were diluted in the same medium to a count of 4 X IO7 do not exceed 48 to 55. At this level of phagocytosis, all bacteria/ml. bacteria become intracellular. Unopsonized Staphylococcus organisms are poorly phago- The tuftsin level of serum activity was assayed as follows. cytized in this system, yielding 0.1 to 0.2, the value obtained 7-Globulin was prepared from freshly obtained inactivated and with opsonized bacteria. Consequently, they are not suitable deopsonized serum by precipitation
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