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[CANCER RESEARCH 33, 1230-1234, June 1973] Phagocytic Activity of the Leukemic and Its Response to the -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 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 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 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 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 morphol During the past few years we have investigated the ogy, along with cytochemical identification of granulocytes and (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, ; L, ; P, ; M, .

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 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 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 with a half-volume of for this assay. All samples of serum used for assaying saturated ammonium sulfate and dialyzed overnight with phagocytic stimulation, as well as the 7-globulin prepared from several changes of 300 ml of phosphate buffer, pH 8.1. Ten mg them, were first inactivated in a water bath for 30 min at 56° of 7-globulin were then digested with 0.5 mg of twice-crystal lized trypsin for 1 hr at 37°.The released tuftsin, if any, was to destroy complement activity. The serum was then deopsonized by exhaustive absorption with washed Staphy extracted with 4 volumes of ethanol. The ethanol extract was lococcus, 3 times each, with 1 mg dry weight per ml in evaporated to dryness and taken up in 0.5 ml of the inactivated serum (5, 6). The reaction was carried out for 30 Krebs-Ringer phagocytosis medium. An aliquot of 0.1 ml,

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1973 American Association for Cancer Research. Constantopoulos, Likhite, Crosby, andNajjar representing 2 mg of the original 7-globulin, was used as the Similar results were obtained in cases of myelofibrosis. The source of tuftsin. All assays were made in duplicate. Several phagocytic index in the basal medium for each of the 6 cases patients were studied on more than 1 occasion, and several was well within normal limits. Again, their cells were not weeks apart, with essentially the same index values. The stimulated either by their autologous serum or synthetic variations encountered were those inherent in the assay. This is tuftsin. exemplified by maximal deviations of ±3in the lower ranges Table 2 includes 2 cases of acute myelomonocytic leukemia. of about 20 and wider variations of ±5in the higher index It is unfortunate that we have not been able to obtain more of values of around 40. such patients because the disease contrasts sharply with the other 2 types of myelogenous dyscrasias. Here the basal RESULTS phagocytic index (reagent control) was significantly elevated much above normal values. Furthermore, both cases showed Phagocytic Activity. Table 2 shows the phagocytic index of maximal stimulation with both autologous serum as well as the white blood cells of the various patients and those of tuftsin. normal control subjects. Cell samples from 5 patients with The results in Table 2 point clearly to a definite deficiency acute granulocytic leukemia showed near normal values in the in the response of the myelocytic phagocyte in acute KRPG basal medium. Only 2 patients, E. D. and A. F., had granulocytic leukemia and in myelofibrosis. While being significantly low phagocytic values. White blood cells obtained capable of near normal basal phagocytosis, it is singularly from all 1 patients did not show any stimulatory response incapable of being stimulated by tuftsin as the tetrapeptide or after the addition of either autologous serum or tuftsin. as an integral part of the autologous serum leukokinin. This

Table 2 Phagocytic index of blood myelocytic cells in leukemic patients before and after stimulation with autologous serum or synthetic tuftsin, compared to normal control subjects Normal subjects represent individuals who were hospitalized for a reason other than blood diseases or lymphatic or bone marrow involvement. Values for phagocytic stimulation were calculated by subtracting the reagent phagocytosis index obtained with the basal KRPG medium from the index obtained in the same medium with added autologous serum or synthetic tuftsin. For details see the text.

Phagocytosis index of cellsCase white blood stimulation A indexwithSerum000(10211000001315202122181923252118202623Tuftsin21001212120001814212323IK2021262220192225

DiagnosisR. alone1414831720132517141822223536202218171920221817192220Serum1314831622142617141822224851404340353843473935394843Tuftsin1615731822142718161721215350414541353941484037384445Phagocytic

granulocyticM.A. Acute C.leukemiaE. D.A. F.J. H.W. K.H. M.G.

B.MyelofibrosisW.U.V.

M.L. P.W. P.F. S.A.

myelomonocyticL.P. Acute S.leukemiaA.

subjectG.F. Normal S.A. L.V.L.V.

N.L. W.P.S.R.

P.N.T.J.P.J.W.K.

N.Reagent

1232 CANCER RESEARCH VOL. 33

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1973 American Association for Cancer Research. Phagocytosis by Leukemic Cells and Tuftsin Effect deficiency may be the result of tuftsin deficiency in the serum show little or no suppression of PMN basal phagocytosis index of these patients. To this end, a study of the serum levels of (reagent controls) compared to normal blood granulocytes. tuftsin activity was undertaken. Only a few gave very low values. However, in all cases, there Tuftsin Activity in Serum. Tuftsin extracts were prepared as was complete resistance to the stimulating effect of their discussed in "Materials and Methods." In previous studies, we autologous serum or the tetrapeptide tuftsin. Furthermore, a showed that dog blood PMN cells gave essentially the same study of tuftsin serum levels in all 13 cases, including both range of response to phagocytic stimulation as did human diseases, showed that tuftsin was very low or not measurable PMN cells (3, 5, 9). The response of dog PMN cells to the in 11 cases and slightly below normal in only 2. prepared tuftsin was therefore used as a measure of tuftsin The 2 patients with acute myelomonocytic leukemia again stimulation. The results are shown in Table 3 which includes were normal with good levels of serum tuftsin activity. normal controls. Four of 7 patients with acute granulocytic leukemia showed DISCUSSION little or no measurable tuftsin levels in their serum. The remaining 3, R. A., E. D., and J. H., had minimal but definite Conflicting results have been reported in studies of serum tuftsin activity. The 6 cases with myelofibrosis also phagocytic activity of the leukemic cell (1,10, 13, 14, 16,17) showed mixed results. Three had practically no serum tuftsin in the presence of autologous or normal serum. Braude et al. while the other 3 showed definite activity, and 2 had values (1) found normal phagocytic activity in 8 of 9 patients slightly lower than normal. suffering from acute myeloid leukemia. Similar results were It is apparent that most but not all cases of either disease obtained in all cases of acute granulocytic leukemia by Silver et al. (16). In contrast, Penny and Galton (10) found Table 3 decreased phagocytic activity in all cases with acute myeloid The level of tuftsin activity in serum j-globulin of leukemic leukemia. They suggested that it is due to "intrinsic defect of patients as compared with normal subjects PMN cells." Sbarra et al. (14) showed normal phagocytic Tuftsin levels were assayed with serum of freshly clotted blood which was inactivated by incubation at 56°for 30 min and absorbed values, although they found "some abnormality in the over-all with washed Staphylococcus. Normal serum controls were obtained phagocytic activity." Recently, Tornyos (17) studied the from patients as described in Table 2. Tuftsin was cleaved by trypsin digestion of -/-globulin that was prepared from these sera. Its activity phagocytic activity of the granulocytes and mononuclear cells was then assayed by the stimulation of phagocytosis, with the use of of the local inflamatory exúdate in 7 patients with acute dog blood PMN cells and opsonized 5. aureus. For details see the text. granulocytic leukemia. He utilized the skin-window technique and found that the phagocytic activity of the cells was Phagocytic significantly low in all cases and suggested that the variable Phagocytic stimulation Case Diagnosis index (A index) observations in previous studies may be related to the difference in techniques used. Finally, Rosner et al. (13) Reagent control 22 reported that from patients with acute granulo cytic leukemia are able to ingest Candida albicans but are R.leuke-M. A. Acute granulocytic incapable of killing the organism. A metabolic defect was C.miaE. D.A. suggested. F.J. In this communication, the study of the phagocytic ability H.W. of the leukemic cell in acute granulocytic or myelomonocytic K.H. types yielded wide variations in the phagocytic capability of M.G. the cells. The series is too small to allow any correlation with B.MyelofibrosisW.U.V.M.L. the severity of the disease. The results are as variable as those obtained by others. However, the only consistent and invariable finding in all 13 cases of both myelogenous P.W. dyscrasias is that the leukemic cell shows complete failure to P.F. S.A. respond to stimulation by synthetic tuftsin or as naturally occurring in serum leukokinin. This is the only known factor myelomonocyticL.P. Acute in inactivated deopsonized serum that stimulates the autol S.leukemiaA. ogous of normal individuals. Its activity parallels in every way the activity of synthetic tuftsin (5,6, 8). subjectG.F. Normal S.A. It has been shown recently that the removal of sialic acid L.V.L.V. from the granulocyte by bacterial neuramini- dase from Vibrio choleras or Clostridium perfringens abolishes N.L.W.P.S.R. the stimulation by tuftsin, leukokinin, or serum (2). It appears that the 1st step in the mechanism of action of tuftsin, i.e., P.N.T.T. binding to membrane sialic, was impaired. Other steps in this activation must precede the invagination and engulfing P.T.W.K. process. If 1 of these steps is missing in the myelocytic cell, then stimulation of phagocytosis beyond a basal level cannot N.3020272329222024342429223448424243384039404145404743408051700212270122620202116181718192318252118 occur. There is another aspect of this problem. Leukemia

JUNE 1973 1233

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1973 American Association for Cancer Research. Constantopoulos, Likhite, Crosby, and Najjar invariably infiltrates the , and all of our patients had phagocytosis should be considered a 3rd possible explanation enlarged . Since tuftsin is made available only in the for the increased susceptibility to infection. presence of this , the levels of serum tuftsin must depend on the relative damage suffered by the spleen. We have already shown that, following splenectomy, tuftsin activity disappears from serum (3, 7,9). Similarly, when the spleen is infarcted, as REFERENCES occurs in sickle cell anemia, or when it is heavily infiltrated by leukemic cells, no tuftsin activity can be found in the serum 1. Braude, A. I., Feltes, J., and Brooks, M. Differences between the (3, 7). It is therefore of interest that over one-half of the total Activities of Mature Granulocytes in Leukemia and Normal Blood. J. Clin. Invest., 33: 1036-1046, 1954. cases showed absent or minimal levels of serum tuftsin. It is reasonable to assume that the spleens of these patients were 2. Constantopoulos, A., and Najjar, V. A. The Role of Sialic Acid in the Phagocytic Activity of Blood Neutrophils. J. Clin. Invest., 5/: heavily infiltrated so that the mechanism by which tuftsin is 21a, 1972. made available was no longer operative. V. M. was the only 3. Constantopoulos, A., Najjar, V. A., and Smith, J. W. Tuftsin patient who was splenectomized, and his lack of serum tuftsin Deficiency. A New Syndrome With Defective Phagocytosis. J. can be explained on that basis. Unlike cases with tuftsin Pediat.,S0: 564-572,1972. deficiency, which have a mutant pep tide that inhibits normal 4. Dameshek, W., and Gunz, F. Leukemia, Ed. 2, pp. 208-344. New tuftsin activity (3), isolated from splenectomized York: Gruñe&Stratton, 1964. individuals (7) and from cases with acute granulocytic 5. Fidalgo, B. V., and Najjar, V. A. The Physiological Role of the leukemia do not show any competitive inhibition of normal Lymphoid System. VI. The Stimulatory Effect of Leucophilic tuftsin. 7-Globulin (Leucokinin) on the Phagocytic Activity of Human Polymorphonuclear Leucocyte. Biochemistry, 6: 3386-3392, The phagocytic activity in the 2 cases of myelomonocytic 1967. leukemia was normal in all respects. Serum levels were normal. 6. Najjar, V. A. The Physiological Role of the Lymphoid System. The cells also responded normally to stimulation by auto- Lymphology, 3: 23-31,1970. logous serum as well as by synthetic tuftsin. These findings 7. Najjar, V. A., and Constantopoulos, A. A New Phagocytosis reflect the responsiveness of the monocyte rather than the Stimulating Tetrapeptide Hormone, Tuftsin and Its Role in high percentage of monocytes present in the blood. In this Diseases. RES J. Reticuloendothelial Soc., 12: 197-215, 1972. connection, 2 cases of myelofibrosis (V. M. and W. P.) showed 8. Najjar, V. A., and Nishioka, K. Tuftsin. A Natural Phagocytosis a similarly high percentage of monocytes but were completely Stimulating Peptide. Nature, 228: 672-673, 1970. without response to stimulation by their serum or synthetic 9. Nishioka, K., Constantopoulos, A., Satoh, P. S., and Najjar, V. A. tuftsin. It must be stressed here that in all cases the same total The Characteristics, Isolation and Synthesis of the Phagocytosis number of cells (2 X IO6) were used in the phagocytosis Stimulating Peptide Tuftsin. Biochem. Biophys. Res. Commun., 47: 172-179,1972. reaction. These 2 cases of myelomonocytic leukemia were 10. Penny, R., and Galton, D. A. G. Studies on Neutrophil Function. included to provide a contrast, and it must not be construed to II. Pathological Aspects. Brit. J. Haematol., 12: 633-645, 1966. mean that the cells of all cases of this disease behave in a 11. Pitocock, J. A., Reinhard, E. H., Justus, B. W., and Mendelson, R. similar manner. S. A Clinical and Pathological Study of Seven Cases of It has been suggested (14, 16) that the increase in Myelofibrosis. Ann. Internal Med., 57: 73-84,1962. susceptibility to infection in leukemic patients is due to a 12. Raab, S. O., Hoeprich, P. D., Wintrobe, M. M., and Cartwright, G. decline in the number of mature and active circulating E. The Clinical Significance of Fever in Acute Leukemia. Blood, phagocytes and not to a decrease in the activity of the mature 16: 1609-1628,1960. phagocyte itself. Our results show a lack of correlation 13. Rosner, F., Valmont, I., Kozinn, P. J., and Caroline, L. Leukocyte Function in Patients with Leukemia. Cancer, 25: 835-842, 1970. between the susceptibility of the cells to phagocytic stimula 14. Sbarra, A. J., Shirley, W., Selvara, R. J., Ouchi, F., and tion and the percentage of blast forms. For example, in acute Rosenbaum, E. The Role of the Phagocyte in Host-Parasite granulocytic leukemia, the number of blasts varied from zero, Interactions. III. The Phagocytic Capabilities of Leukocytes from in 2 cases with remission, to 98% in relapsed patients. Myeloproliferative and Other Neoplastic Disorders. Cancer Res., However, the cells in all these cases showed no stimulatory 25: 1199-1206, 1965. activity with autologous serum or tuftsin. It is also possible 15. Silver, R. F. Infections, Fever and Host Resistance in Neoplastic that reduced leukocyte viability and intracellular killing of Diseases. J. Chronic Diseases, 16: 677-701, 1963. bacteria in some patients might be a factor (13). 16. Silver, R. F., Beai, G. A., Schneiderman, M. A., and McCullough, The data presented here cannot distinguish between these 2 N. B. The Role of Mature Neutrophils in Bacterial Infections in Acute Leukemia. Blood, 12: 814-821,1957. alternatives. There is, however, a clear indication that the cell 17. Tornyos, K. Phagocytic Activity of Cells of the Inflammatory cannot be stimulated to normal levels of phagocytic activity Exúdate in Human Leukemia. Cancer Res., 27: 1756-1760, 1967. by added synthetic tuftsin or by the tuftsin present in 18. Viola, M. V. Acute Leukemia and Infection. J. Am. Med. Assoc., autologous serum. Both W. U. and F. S. showed near 201: 923-926,1967. low-normal values of serum tuftsin activity, yet their serum 19. Yam, L. T., Li, C. Y., and Crosby, W. H. Cytochemical failed to stimulate their own cells. Thus, this failure of the Identification of Monocytes and Granulocytes. Am. J. Clin. phagocyte to be stimulated beyond a certain level of Pathol., 55: 283-290, 1971.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1973 American Association for Cancer Research. Phagocytic Activity of the Leukemic Cell and Its Response to the Phagocytosis-stimulating Tetrapeptide, Tuftsin

Andreas Constantopoulos, Vilas Likhite, William H. Crosby, et al.

Cancer Res 1973;33:1230-1234.

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