Europ. J. clin Invest. 5 , 215-219 (1975)
Quantitative Iodination of Human Blood Polymorphonuclear Leukocytes J. Hakim, E. Cramer, P. Boivin, H. Troube, and J. Boucherot Centre de Recherches sur les Enzymopathies de l'hssociation Claude Bernard et Unit& 2 4 de 1'INSERM; H6pital Beaujon, 100 boulevard du G&n&ral Leclerc, 9 2 1 1 0 Clichy, France Received: March 1 5 , 1974, and in revised form: August 12, 1974
Abstract. It was shown by Pincus and Klebanoff that a correlation existed between leukocytic iodination measured i n vivo and microbicidal leukocytic activity. We have analysed the results of this test in relation to time and in the presence of variable quantities of polymorphonuclear leukocytes (PMN). The values observed per time and PMN unit proved to be equivalent in the presence of 2.5 x lo5 PMN or 5.0 x lo5 PMN per 0.5 ml of incubation medium, measured after 10, 2 0 and 30 minutes or in the presence of I .O x lo6 PMN, measured after 10 minutes. That is to say iodination is proportional to leukocyte concentration and incubation time. Increase of either the quantity of cells or the incubation time, beyond the area we defined, reduce iodination per cell and per unit of time. Concerning the patients with an insufficient iodination, we have studied 2 parameters in the presence of 5.0 x lo5 PMN:I) initial iodination measured after 10 and 20 minutes and 2) stability of iodination measured after 60 minutes. These two parameters were equally affected in two cases with myelofibrosis, 3 patients with acquired refractory anaemia, one with chronic lymphoid leukaemia, one with erythroleukaemia, one with hairy cell leukaemia, one with systemic mastocytosis and almost complete myeloperoxidase deficiency, one with sickle cell disease, two with liver diseases and two with chronic myeloid leukaemia. The iodination at the 60th minute was more affected than at the 10th minute with a patient with myelofibrosis and 4 other patients with acquired refractory anaemias. The significance of these differences is not well understood; however the meaning of the decrease in the iodination of whatever type is that a PMN anomaly exists directly related to the myeloperoxidase I1202 halogenation system, or to one of the stages of engulfment and/or metabolic events preceeding it and leading to the production of H202. This test, with the alterations we introduced, is suggested as a test for detection of functional PMN abnormalities.
Key Ltords: Iodination, polymorphonuclear blood cells, leukocytic metabolism, health, disease, humans.
gical conditions. However the complete functional study of the PMN is difficult, time consuming and still reserved to a few specialized laboratories. Pincus and Klebanoff (11) have described a A screening test of PMN functional abnormality quantitative leukocyte iodination test measuring would allow a selection of cases to be studied in uitro the conversion of iodide to a trichloroextensively for PMN functional defects. As to the acetic acid precipitable form by phagocytosing leukocytic iodination test, it would measure the human leukocytes. Iodination was found to be reterminal enzymatic stage of a leukocytic microlated to a certain extent to the leukocytic microbicidal activity. The authors therefore recommended bicidal system leading to the production of hydrogen peroxide (H202) and to its uses to iodinate that this technically simple test should become an addition to ,the other techniques for the evaluation the proteins (4) in a covalent way by myeloperoxiof the leukocytic function. Hereditary ( 2 , 6 , 9) or dase (6). In fact the iodination phenomenon is a very complex one; its measurement simultaneously acquired (3) conditions obviously exist in which reflects the ability of the leukocytes to engulf, polymorphonuclear cells (PMN) are functionally intheir sensitivity to metabolic activation by ensufficient. That might account for the multipligulfment, the production of superoxide and peroxide, cation and dissemination of microorganisms (bacthe conversion of superoxide to peroxide, the esteria, fungi and viruses) responsible for severe cape of the latter substance from the glutathione infections in man. Moreover, one of the PMN cell and the catalase systems, the amount of myelopefunctions is the elimination of foreign particles roxidase and the rates of degranulation (6, 14). other than microorganisms such as immune complexes Moreover if iodination was first considered as and perhaps abnormal cells. Thus more and more an important system of bactericidal (S), candiciattention is focused upon the study of the PMN dal (8) and viricidal ( 1 ) activities in V i V O ; at function and its measurement in different patholo-
J. Hakim e t a l . : Quantitative Iodination of Human Blood
tube, This cellular suspension was gently mixed with an equal volume of 0.87 % ammonium chloride to haemolyse the remaining red blood cells. After centrifugation for 8 minutes at 200 g the leukocytes were washed twice with W e b s Ringer Phosphate buffer, pH 7 . 4 without calcium (KRP); a differential count was made and the final suspension was adjusted in the same buffer bo 5 x lo7 PMN + monocytes per ml or, most often, to I x 107 PMN + monocytes per ml. Dilutions of the final leukocyte suspension were made in the same buffer when necessary. The terminal cellular suspension contained 80 to 90 X neutrophil PMN. The cell viability was assessed by the trypan blue test. Zymosan (Mann. Res. Lab. New York) was suspended in the KRP buffer (5 mg/ml) and homogenized for one minute in a Potter Elvehjem Teflon and glass homogenizer. Human serum from healthy adult male volunteers whose blood group was AB (S-AB) frozen in fractions of 1 ml, was used immediately after thawing. SodiumlP5 iodide, carrier free, in 0.01 M sodium hydroxide (Commissariat de 1'Energie Atomique, Saclay, France) was used as a tracer for non radioactive sodium iodide ( 1 x M) in the KRP buffer. Measurement of iodination was performed according to Pincus and Klebanoff ( 1 1 ) . Based on the studies to be presented in this paper the final reaction mixture consisted of, except when stated, 2.5 x lo5 to 5 x lo6 PMN, 10 X of S-AB, 10 nanomoles of sodium iodide (about 0.2 uCI of , 0.5 mg of zymosan approximately 5 x lo7 particles and KRP buffer to a total volume of 0.5 ml. Incubation was at 37OC in plastic test tubes 12 mm in diameter oscillating 5 4 times per minute in a Dubnoff shaker. Tyo identical tubes were run simMaterial and Methods ultaneously. All the ingredients of the reaction were pre-heated to 37OC and the chronometer was The control group consisted of forty normal released with the addition of the cellular sussubjects, members of the medical and paramedical pension which was added last. The reaction was staff of Beaujon's hospital. The pathological substopped at the loth, 20th, 30th and 60th minutes jects whose iodination results are reported here with 0.1 ml of 0.1 M sodium thiosulphate and lml were selected (within a set of systematic studies of 10 % cold trichloroacetic acid ( 1 1 ) ; the pre0 of PMN function in different pathological cases) cipitate was collected by centrifugation ( 4 C) at because of an inadequate iodination function. The 2,000 g for 5 minutes and washed four times with group comprised 3 agnogenic metaplasias of the 1 ml of cold 10 X trichloroacetic acid. The radiospleen with myelofibrosis, 7 acquired refractory activity of the precipitate was counted in a anaemias, a chronic lymphoid leukaemia, two chronic Picker Nuclear gamma counter (Twinscaler 11). A myeloid leukaemias, an erythroleukaenia, a hairy blank containing all the reagents, except the cell leukaemia, a systemic mastocytosis with a mye- leukocytes was included and its value subtracted loperoxidase deficiency, a sickle cell disease, an from the experimental value before calculating the alcoholic cirrhosis of the liver and a Wilson disiodination. The results were expressed in nanoease. All of these subjects were free from infecmoles of iodide converted to a trichloroacetic tion when studied. acid precipitable form per lo7 PMN per hour. The The isolation of leukocytes was carried out effect of lymphocytes was neglected after a prefrom venous blood collected in plastic tubes conliminary study showing that the iodination due to taining lithium heparinate (10 units per ml of a suspension of lymphocytes was practically insigblood). The blood was rapidly transferred to a nificant tube containing sodium ethylene diamine tetra acetate (FDTA : 1.4 mg per ml of blood). 20 ml of blood were mixed with 10 ml of a 2 % Dextran T 5 0 0 solResults ution (Pharmacia, Uppsala, Sweden) in 0.9 % sodium chloride and allowed to stand for 30 to 45 minutes I ) Control Subjects at room temperature (20 to 24OC). The leukocytes collected from the supernatant, after sedimentation A. The effect of the incubation time and of the of the majority of the red blood cells, were trans- PMN concentration on the iodination reaction, calferred into a siliconized glass conical centrifuge culated for all the experimental conditions per
present the iodination test should only be considered as reflecting the activity of the "myeloperoxidase-H202halogen systems". The biochemical mechanism by which it kills the microorganism is certainly more complex (7, 12) than initially described (4). The iodination test measures a very complex variable and iodination may not be implied in i n uivo bactericidal activity. Nevertheless the results of the measurement of this metabolic event which is a final stage of an important chain conditioned by engulfment as well as by all the metabolic steps leading to it, may be accepted as a good reflection of the functioning of the whole system. It is obvious that decreased iodination unquestionably calls for a complete PMN functional study in order to focus upon the stage or the stages primarily responsible for the anomaly. In the present study the results of the iodination test made on the leukocytes of normal and pathological subjects are reported. We have insisted upon some methodological points and analyzed some of the characteristics of the reaction because of discrepancies between the results reported by Pincus and Klebanoff (11) and those reported by Olsson e t aZ. (10). Considering this test as the first step in the detection of a PMN functional anomaly we tried to define the conditions of highest sensitivity. For the reported pathological cases we have deliberately left aside the analysis of the different factors (engulfment and/or metabolic events) that might be responsible for the observed decrease of iodination.
J. Hakimet al. : Quantitative Iodination of Human Blood
lo7 PMN and per hour, is reported on Table I . The iodination measured at the 20th and 30th minute, was proportional to the PMN concentration over a range of 2 . 5 to 5 x l o 5 PMN per reaction mixture. A PMN concentration higher than 5 x lo5 resulted in decreased iodination per cell. This decrease was more pronounced as the PMN concentration increased. The iodination measured at the 60th minute increased per cell when the number of polynuclears was decreased. The iodination measured at the 10th minute was proportional to PMN concentration over a range of 2.5, to 1 x lo6 PMN. A concentration higher than 1.0 x l o 6 PMN resulted in decreased iodination per cell. Thus, iodination was proportional to PMN concentration as well as incubation time in the area defined by dashed lines on Table 1 . An increase in cell concentration of the incubation mixture outside the area we have defined, caused on the one hand a decrease in the initial iodination per cell and, on the other hand, an accentuation of the decrease in iodination per cell in relation to time. B. In the presence of a low concentration of PMN (2.5 x IO5/0.5 ml) the dilution of the zymosan in the incubation medium to 1/20th resulted in lower iodination per cell in the 10th minute (Table 2) than that observed with undiluted zymosan (Table I ) . The iodination was however of the same order as that measured in the presence of 5 x lo6 PMN and undiluted zymosan. The ratio of the number of PMN and zymosan particles in the incubation medium was the same in both cases. Nevertheless, when the iodination was measured over a longer period of time, it decreased per unit of time with the highest concentration of PMN while it remained stable with the lowest concentration. In our standard mixture (Table 1 ) the iodination was proportional to time up to 30 minutes and maximal with regard to a ratio of zymosan particles to PMN equal to or higher than
Table 1 . Iodination (nanomoles/107 PMN/60 minutes)' Number of PMN/ 0.5 ml of incubation medium 5 2.5 x 10
Incubation Time (min.) 10'
- --- --- - -- ,
5 5.0 x 10
1.0 x 10
1I 16.35 r----I 15.07 11.71
2.5 x 10
6 5.0 x 10 (8)
12.24 C2.57 8.41 '2.20
2 1 .48
'Mean 2 standard deviation with number of controls studied in parentheses. The reaction mixture contained 0.5 mg of zymosan and 10 nanomoles of iodide in a total volume of 0.5 ml and the other components specified in material and methods. The experimental results are expressed for all the measurements per lo7 PMN and par hour. Table 2. Iodination (nanomoles Number of PMN/number of zymosan in 0.5 ml incubation medium 5
6 IOPMN --1 7 10 part. 10
lo7 PMN/60 minutes)t
Incubation time (min.) 10'
C. The PMN iodination 2t various concentrations 5 7.65 7.54 7.88 7.08 2.5 x 10 PMN of iodide is shown in Table 3. An increased iod--1 6 ination was observed which reached a maximum for 2.5 x 10 part. 10 21.28 t1.68 21.98 22.27 50 nanomoles of iodide/0.5 ml incubation mixture. (7) The iodination decreased slightly with higher concentrations of iodide. 'Mean 2 standard deviation with number of controls D. Our results of iodination measurement in the absence of serum or of zymosan particles in the in- studied in parentheses. The experimental results are expressed in all the measurements per lo7 PMN cubation medium confirmed those o f Pincus and and per hour. Klebanoff ( 1 1 ) showing that these factors are necess ary for iodination. Moreover, like these authors, we found that an almost complete inhibition of iod- Table 3. Iodination with different amounts of iodide, iodide concentration (pM) ination occurs in the presence of cyanide ( I x M) 20 40 60 80 100 120 200 E. Five repeated measurements of iodination made approximately at one month intervals on six differ100 130 160 179 196 178 162 ent controls (with the conditions stated in Table 1 Mean 1 SD by dashed lines) did not differ for each control (8) t22.5 27.9 218.1 225.9 232.0 235.1 230.9 by more than 6 % of the mean of the five results.
2) Pathological Subjects
We are not reporting here the results of systematic surveys which are being carried out in different pathological conditions but only selected '
Iodination was measured in 0.5 ml incubation medium at the 10th min. in the presence of 5 x lo5 PMN. Results are expressed as percentage of iodination measured with 10 nanomoles of iodide (20pM). The number of controls studied is in parentheses.
J. Hakim e t a l . : Quantitative Iodination of Human Blood
cases found deficient in iodination (Table 4 ) . The iodination was measured in the presence of 5.0 x l o 5 PMN and 10 nanomoles iodide, Three subjects with agnogenic metaplasia of the spleen with myelofibnosis were found deficient. The first had a very pronounced deficiency and the second a moderate one. With the third the deficiency only appeared clearly when the time of measurement was 60 minutes. Seven subjects with acquired refractory anaemia, were found deficient: with two of them (patients 1 and 2 ) the deficiency was very sharp; with four of them (patients 3 , 4 , 5 and 6 ) the deficiency was less pronounced than with the first two and more clearly visible after a measurement at 6 0 minutes than at 10 minutes. With the seventh, who was affected by a Pelger Huet anomaly of his PMN, the iodination deficiency was sharper at the 60th minUte than at the 10th minute. Three subjects with respectively, a chronic lymphoid leukaemia, an acute erythroleukaemia and a hairy cell leukaemia, were in a very stable way, deficient as a function of time. Three subjects with respectively heterozygous sickle cell disease, alcoholic cirrhosis and Wilson disease had a relatively stable and moderate iodination deficiency as a function of time. A patient affected by a systemic mastocytosis with an almost complete lack of myeloperoxidase (less than 10 X of the controls) had an important deficiency of iodination. In two subjects with hyperleukocytic myeloid chronic leukaemia, the iodination was found sharply decreased.
Table 4 . Iodination Incubation time (min.) 10' 20' 30' 60' 71
22.6 55.5 70.1
24.5 54.3 66.5
22.7 52.8 57.6
24.3 53.3 46.2
35.4 26.0 64.4 53.2 99.3 72.6 43.7
34.6 27.2 55.7 52.7 98.0 30.8
36.2 28.0 51.8 54.8 65.4 54.0 28.2
40.0 29.2 40.2 53.8 59.0 48.5 25.2
Chronic lymphoid leukaemia
Hairy cell leukaemia
Sickle cell anaemia
Lowest value observed in the controls' Agnogenic metaplasia of the spleen with mye1 lofibrosis 2 3
Acquired refractory anaemia
Chronic myeloid leukaemia
Our results, on normal subjects, are in partial disagreement with those of Pincus and Klebanoff ( 1 1 ) for unknown reasons. These authors reported that iodination was proportional to the number of leukocytes over a range of 0.25 to 2 x lo7 cells for an incubation medium cf 0 . 5 ml'at the 60th minute. We found that, for measurements made at the 60th minute, the iodination per cell increased when the PMN concentration decreased from 5 x lo6 to 2 . 5 x l o 5 cells. For a PMN concentration of 2 . 5 x lo5 and 5 x lo5 we found iodination to be proportional to time up to 3 0 minutes. The decreased iodination per cell in the presence of a high concentration of PMN indicates a crowding effect and/or a relative defect of zymosan. The ratio of the number of zymosan particles to the number of incubated PMN seems to have to be higher than 100 for the iodination to remain maximal and stable until the 30th minute. We observed as did Simmons and Karnowsky ( 1 3 ) that increased levels of iodide in the medium increased iodination. However in disagreement with these authors we found that above a concentration of iodide of 1 x IO+M, iodination did not increase further. It even decreased slightly. However these authors studied the effect of raising the iodide concentration with a different system in which adhesivity of PMN was lost in the presence of excess iodide thus selecting the PMN studied. Our system avoids.this problem. Thus iodide concentration seems to be limiting but only to a certain extent.
2 3 4 5 6 7
Iodination was measured in 0 . 5 ml incubation medium ir the presence of 5 x lo5 PMN, 0.5 mg zymosan and 10 nanomoles of iodide. The experimental results were expressed as percentage of the mean control levels (Tab1 I ) . The values are the mean of 2 experiments made usually on two consecutive days with duplicate tubes for each. Our results in normal subjects are in close agreement with those of Olsson e t a2. (10). Thus, the measurement of an initial highest speed of iodination should, in our opinion, be made, in the presence of less than 5 x lo5 PMN/O.5 ml over 10 to 3 0 minutes, in the presence of 50 nanomoles iodide and of one hundred or more zymosan particles per cell. In pathology the iodination measured over a short period of time (10 minutes) shows the initial speed of iodination; while the measurement over 60 minutes shows its stability. The standard conditions that we have chosen for the study of patients included a number of cells of 5 x lo5 PMN in 0.5 ml and measurements of iodination made during 10, 2 0 and 60 minutes in the presence of 0 . 5 mg of zymosan and of 10 nanomoles of iodide. The low concentration of iodide was chosen to allow the comparison with the already published data. The systematic study of iodination by the PMN has shown us that acquired insufficiencies of this function were not exceptional, especially in
J . Hakim e t aZ.: Quantitative Iodination of Human Blood
patients with myelofibrosis, refractory anaemias, leukaemias and liver diseases. In the case of systemic mastocytosis, a family study was not made and it is at present difficult to say if this deficiency is a part of the illness or if it is only a coincidence. In most of our reported cases the deficiencies are clearly observed at the 10th minute; nevertheless, some of them seem to be more pronounced at the 60th minute and in one case, the deficiency appeared only at the 60th minute. The meaning of this phenomenon is unclear. The existence of insufficient iodination does not necessarily imply that it is an abnormality initially located in the "myeloperoxidase H202 iodide" system but it can be in keeping with one of the stages preceding and influencing the iodination that is to say engulfment and/or post engulfment metabolic events. So, this test, made individually, appears to be a relatively simple technique to show a PMN functional trouble. In pathology it is important to study fully the granulocytic function when defective iodination is detected to locate the abnormality exactly.
References I . Belding, M.E., Klebanoff, S.J., Ray, C.G.:
Peroxidase mediated viricidal systems, Science 167, 195 (1970) 2. Douglas, S.D.: Analytical review: Disorders of phagocytic function. Blood 35, 851 (1970) 3. Hakim, J., Boivin, P., Boucherot, J., Troube, H.: Granulocytopathies acquises. Nouv. Rev. franc. H'emat. 14, 284 (1974) 4 . Klebanoff , S . J . : Iodination of bacteria: a bactericidal mechanism. J. exp. Med. 126, 1063 ( 1 967)
5. Klebanoff, S.J.: The antimicrobial activity of catalase at acid pH. Proc. SOC. exp. Biol. (N.Y.) 132, 571 (1969) 6. Klebanoff, S.J.: Intraleukocyte microbicidal defects. Ann. Rev. Med. 22, 39 (1971) 7. 'Klebanoff',S.J., Hamon, C.B.: Role of myeloperoxydase mediated antimicrobial systems in intact leukocytes. J. reticuloendoth. SOC. 12, 170 (1972) 8. Lehrer, R.I.: Antifungal effects of peroxidase systems. J. Bact. 99, 361 (1969) 9. Nathan, D.G., Baehner, R.L.: Disorders of phagocytic cell function. In: Progress in Haematology. E.B. Brown and C.V. Moore (Eds.), VII, p. 235. New York-London: Grune and Stratton 1971 10. Olsson, I., Olofsson, T., Odeberg, H.: Myeloperoxidase-mediated iodination in granulocytes. Scand. J. Haemat. 19, 483 (1973) 1 1 . Pincus, S.H., Klebanoff, S.J.: Quantitative leukocyte iodination. New Engl. J. Med. 284, 744 (1971) 12. Sbarra, A.J., Paul, B.B., Jacobs, A.A., Strauss, R.R., Mitchell, G.W., Jr.: Role of the phagocyte in host-parasite interactions XXXVIII. Metabolic activities of the phagocyte as related to antimicrobial action. J. reticuloendoth. SOC. 12, 109 (1972) 13. Simmons, S.R., Karnovsky, M.L.: Iodinating ability of various leukocytes and their bactericidal activity. J. exp. Med. 138, 44 (1973) 14. Stossel, T.P.: Phagocytosis (Second of three parts). New Engl. J. Med. 290, 774 (1974)
Dr. J. Hakim Centre de Recherches sur les Enzymopathies Hzpital Beaujon Bd. du G'en'eral Leclerc 100 F-92110 Clichy France