Immunology 1977 32 491
Quantitative phagocytosis by human polymorphonuclear leucocytes USE OF RADIOLABELLED EMULSIONS TO MEASURE THE RATE OF PHAGOCYTOSIS
A. FORSGREN, D. SCHMELING* & 0. ZETTERVALL Departments of Clinical Bacteriology and Internal Medicine, University ofLund, General Hospital, Malmo, Sweden
Received 1 July 1976; acceptedfor publication 27 August 1976
opsonized by serum factors including antibodies and complement. Numerous attempts have been made to relate abnormalities in function of the neutrophil granulocytes and opsonins with the development of bacterial infection. However, difficulties in methods and lack of standardization of techniques have resulted in divergent interpretations of the experimental results. Phagocytosis can be assayed in vitro by various methods including microscopic examination of stained smears (Hanks, 1940) and methods involving cultivation of nonphagocytosed or surviving bacteria (Maal0e, 1946). Other widely used methods utilize the measurement of radioactive particles (Cohn, 1963). More recently, a phagocytosis method involving spectrophotometric determination of ingested oil has been described (Stossel, Alper & Rosen, 1973). This paper describes a method which is a modification of the spectrophotometric method described by Stossel et al. (1973) based on the quantification of ingestion of lipopolysaccharide (LPS) coated oil particles which were radioactively labelled. By this method it is possible with as little as 50,000-1 million polymorphonuclear leucocytes or 10 ,ul of serum to quantify precisely and rapidly the ingestion rate of phagocytes and the opsonic activity of serum.
Summary. A new micro-method for the quantitative measurement of phagocytosis by neutrophils is described. The material used for phagocytosis consists of a radioactive oil emulsion coated with E. coli lipopolysaccharide. Uptake of radioactive material is a function of cell number, duration of incubation, dilution of serum used for opsonization, content of lipopolysaccharide and concentration of emulsion. This method can be used to quantify rapidly and precisely phagocytosis rates of as few as 5 x 104-106 polymorphonuclear leucocytes and the opsonic activity of 10 ju1 serum.
INTRODUCTION
Phagocytosis and subsequent killing of microorganisms are major defence mechanisms against pyogenic infections. However, many bacteria are poorly ingested by phagocytic cells unless first * Present address: Department of Pediatrics, University of Minnesota, Medical School, Minneapolis, Minnesota 55455, U.S.A. Correspondence: Professor A. Forsgren, Department of Clinical Bacteriology, University of Lund, Malmb General Hospital, S-214 01 Malmo, Sweden.
491
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A. Forsgren, D. Schmeling & 0. Zettervall
MATERIALS AND METHODS Leucocytes and sera Human peripheral leucocytes from healthy laboratory personnel were prepared by dextran sedimentation of heparinized venous blood and three times in Krebs-Ringer phosphate medium pH 7 4 and finally suspended in the same medium with the addition of 3 per cent bovine serum albumin (Poviet Producten N.V., Amsterdam) at a concentration of 5 x 107 polymorphonuclear leucocytes per ml in most experiments. Serum was collected from clotted human blood by standard laboratory techniques. The sera were stored for not more than 2-3 h at 40 or frozen immediately at -70°.
Preparation of antigen Lipopolysaccharide was extracted from Escherichia coli 0: 118 using a modification of the phenol-watermethod of Westphal, Luderitz & Bister (1955). After dialysis of the water phase against distilled water and lyophilization, the material was dissolved in a small volume of distilled water and precipitated once with ethanol in the presence of a small amount of sodium acetate. Centrifugation at 100,000 g was performed several times until free of material absorbing at 260 mp. Preparation of radioactive oil Commercial corn oil was radiolabelled with Potassium Iodide (1251) (The Radiochemical Centre, Amersham) by a chloramine T-method. One hundred pl of corn oil was dissolved in 300 p1 of chloroform. Ten pl of distilled water containing 10 ,ug of KI and 1 mCi of 25I1 followed by 40 pg of chloramine-T in 20 pul of 0 05 M phosphate buffer, pH 7 5 were added. After the development of a colour change, indicating free iodine in solution, the tube contents were mixed by shaking until the iodine colour was evenly distributed throughout the organic phase. The reaction was allowed to proceed for 1 h and then stopped by the addition of 80 pg of sodium meta bisulphite in 20 ,ul of water. Unreacted iodine was extracted from the mixture by shaking with several 5-ml portions of water. Twenty per cent of the added radioactivity was recovered in the first 5 ml of water whereafter no significant radioactive content was noted in the water. The chloroform was removed from the organic phase with a stream of nitrogen. The labelling was
performed by Mr Ingvar Larsson, The Diagnostic Isotope Laboratory, General Hospital, Malmo. Approximately 1 per cent 125I-labelled corn oil was added to, and carefully mixed with, nonradioactive diisodecylphthalate (ICN Pharmaceuticals Incorporated, Life Sciences Group, Plainview, New York) to which Red Oil 0 (Sigma Chemical Corporation) had been added (Stossel et al., 1972). Preparation of emulsions Emulsions of nonradioactive antigen and radioactive oil were prepared as follows: E. coli lipopolysaccharide was normally suspended at a concentration of 10 mg per ml in Krebs-Ringer phosphate medium in a plastic tube and dispersed by sonication for 15 seconds (MSE Ultrasonic Disintegrator, 3 mm microtip, Measuring and Scientific Equipment Ltd,
London). One volume of oil was then layered over three volumes of lipopolysaccharide suspension and the oil was emulsified into droplets by sonication according to a standardized technique. Total volume of the emulsion was usually 100,ul but occasionally a large amount of emulsion was prepared, divided into aliquots of 100 ,ul and frozen at -20°. Opsonization of the emulsion Test serum used for opsonization either undiluted or in two-fold serial dilutions in Krebs-Ringer phosphate medium containing 3 per cent albumin was incubated during rotation with an equal amount of oil emulsion at 370 for 15 min. The amount of serum used for testing of individual sera was usually 10 ,pl which was adequate for a duplicate or triplicate phagocytosis test. For other experiments larger amounts of emulsion were opsonized and divided into aliquots.
Phagocytosis procedure To siliconized round-bottomed glass tubes was added 10 p1 of a leucocyte suspension containing 101-4 x 106 polymorphonuclear leucocytes in KrebsRinger phosphate medium or in 1 MM N-ethylmaleimide (BDH Chemicals Ltd, Poole) dissolved in the same buffer. In most experiments 5 x 105 neutrophils were used. The tubes were shaken in a water bath at 370 for 5 min. Five ul of opsonized or nonopsonized emulsion was added to each tube in an experimental set in intervals of 15 s to allow for pipetting. After 5 additional min of shaking at 37° 3 ml of ice-cold 1 mM N-ethylmaleimide in 015 M
Phagocytosis of radioactive oil emulsion sodium chloride was added in 15-s intervals in the same order. The leucocytes were obtained as a pellet by centrifugation at 500 g for 10 min and the supernatant containing uningested emulsion was removed with a Pasteur-pipette and the inside of the tube was carefully wiped with a cotton swab. The cell pellet was resuspended in 3 ml of fresh saline and after centrifugation and discarding the supernatant the tube walls were wiped again and finally the 25Iradioactivity was measured in an automatic gamma counter (LKB-Wallac 80,000). All tests were done in duplicate or triplicate. RESULTS
Influence of medium on phagocytosis Table 1 shows the influence of albumin when added to a standard incubation medium used for phagocytosis experiments. Radioactivity in the cell pellet after incubation was essentially independent of the presence of albumin when an opsonized emulsion was used. However, as also indicated in the figure, an unexpectedly high radioactivity was measured in the absence of albumin when nonopsonized emulsion was tested. In some experiments an even higher radioactivity was obtained in the cell pellet when
nonopsonized emulsion was used in comparison to that recorded when the emulsion had been opsonized by serum in moderate dilutions. This shows that in the absence of albumin a cell-bound radioactivity which is nonrelated to opsonization of the oil particles can be obtained. However, as also shown in Table 1 this undesirable high uptake of nonopsonized particles can be nearly completely avoided by the addition of albumin to the incubation medium. Table 1 shows a low radioactivity in tubes without cells indicating a low degree of adherence of oil particles to the glass tube walls. However, even in the experiments without cells the radioactivity was lower when albumin was present and the radioactivity in the tubes represented 0 5 per cent of added radioactivity as compared to 1 per cent in the absence of albumin. In addition Table 1 reveals the reproducibility of the test. The coefficient of variation for values determined at 6 min in all experiments was found to be 7 per cent. Other phagocytosis experiments were performed with 0-1 per cent gelatin added to the incubation medium as previously described by Ward & Zvaifler (1973). Gelatin did not, however, significantly deter the adherence of nonopsonized particles to leucocytes. The presence of gelatin had no marked effect on phagocytosis of opsonized particles.
Table 1. Influence of albumin on cell-bound radioactivity
Neutrophils and opsonized emulsion
Albumin in medium
Radioactivity in pellet (c.p.m.)
-
2829 2858 2844 (Mean) 2590 2880 2735 (Mean) 1557 1476 1517 (Mean) 161 176 168 (Mean) 78 89 84 (Mean) 44 34 39 (Mean)
+
Neutrophils and nonopsonized emulsion
-
+ No neutrophils and nonopsonized emulsion
493
-
+
494
E
A. Forsgren, D. Schmeling & 0. Zettervall
and not only an attachment of opsonized particles to polymorphonuclear leucocytes.
800(
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Influence of time on phagocytosis When a constant amount of polymorphonuclear leucocytes (5 x 1O5) was incubated with a preopsonized lipopolysaccharide oil emulsion the uptake increased rapidly during the first two minutes whereafter the rate of ingestion levelled off (Fig. 2).
600( _L
z
0
400(
-c :3
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200(
9
=
-*-i
a
,,
05 1.0 1.5 2-0 Number of polymorphonucleor leucocytes (x 106)
4.0
Figure 1. Phagocytosis as a function of cell concentration. Normal human leucocytes in a 10 p1 Krebs-Ringer phosphate medium containing 3 per cent albumin were incubated for 6 min at 370 with a constant amount of emulsion containing lipopolysaccharide (10 mg/ml) and a mixture of '251-labelled corn oil and diisodecyl phthalate. The emulsion was opsonized with undiluted serum and added to tubes in volumes of 5 pi. Each point is the mean of duplicate values. The radioactivity obtained in the cell pellet when 0-5 x 106 polymorphonuclear leucocytes were suspended in 1 mM N-ethylmaleimide is indicated by a hatched column.
c3000 -
E
i
Ci
I
2
2000-
-
1000 -
Time (min)
Influence of cell concentration on phagocytosis
Fig. 1 shows a phagocytosis experiment with 5 pl opsonized lipopolysaccharide oil emulsion and polymorphonuclear leucocytes in different numbers ranging from 10,000-4 million corresponding to 1 x 106-4 x 108 cells per ml. The uptake during the first 6 min of incubation was directly proportional to cell number in the range from 10 thousand-1 million, being 3 and 45 per cent of added emulsion respectively. A still greater uptake was obtained when higher cell numbers were used. However, at 1 x 106 cells a plateau was reached where 60 per cent of added emulsion was phagocytosed and 4 x 106 cells gave no additional uptake. The figure also indicates the influence of N-ethylmaleimide on radioactivity in the cell pellet. Cells suspended in buffer containing 1 mM N-ethylmaleimide gave a low degree of radioactivity in the cell pellet. When 5 x 105 cells (a cell number used in all other tests) were suspended in N-ethylmaleimide only 3 per cent of total added radioactivity was recovered in comparison to 23 per cent for the same number of cells suspended in buffer without N-ethylmaleimide indicating that the radioactivity of the pellet was reflecting an active phagocytio process of the cells
Figure 2. Phagocytosis as a function of time. Ten p1 of normal human leucocytes (0-5 x 106) were incubated with 5 jul of the opsonized emulsion described in Fig. I (closed circles). For comparison the rate of phagocytosis of a nonopsonized emulsion is included in the figure (open circles).
After 6 min a plateau was reached with an uptake of 23 per cent of added radioactivity and no further uptake was obtained. Uptake of nonopsonized particles was strikingly lower than that with opsonized particles. A plateau was also reached with nonopsonized particles the level of this being 11 per cent of the values obtained with opsonins present. Influence of opsonization on phagocytosis Fig. 3 reveals that the opsonic activity of human serum as determined in the standard assay system was limiting and was proportional to serum concentration in the range of 12 5-100 per cent corresponding to serum dilutions 1:8 and undiluted. The percentage uptake of added radioactivity was 23, 15, 1 1, and 8 for serum dilutions undiluted 1: 2, 1:4, and 1: 8, respectively. When lower concentrations of serum were used for opsonization a sharply decreasing rate of phagocytosis was obtained. At a serum
495
Phagocytosis of radioactive oil emulsion tD
E w
3000
{
E
E >
3000
2000
_
2000
-0
1000 _
1000
/
0
0
12!5 25
50
100
Serum (per cent)
Figure 3. Phagocytosis as a function of opsonization. Ten microlitres of normal human leucocytes (5 x 107 per ml) were incubated with a lipopolysaccharide-oil emulsion preopsonized with varying concentrations of serum in a final volume of 5 j1u. Nonopsonized emulsion was included as a control. The radioactivity obtained when emulsion was opsonized with heated serum is indicated by a hatched column.
Figure 4. Phagocytosis as a function of content of lipopolysaccharide in suspension used for preparation of emulsion. Normal human leucocytes in 10 p1 medium incubated for 6 min at 370 with 5 p1 of emulsified oil coated with varying amounts of lipopolysaccharide. Each point is the mean of duplicate values.
(0
200C Id
concentration of approximately 3 per cent (serum dilution: 1:32) no appreciable difference was obtained compared to the control where no serum was used. Fig. 3 also shows the low degree of radioactivity obtained in the cell pellet when an emulsion opsonized with heat-inactivated serum (560, 30 min) was used. Influence of emulsion content and concentration on phagocytosis As shown in Fig. 4 maximal uptake was obtained when E. coli lipopolysaccharide in a concentration of 10 mg per ml was used for preparation of the emulsion, the uptake at 5 mg per ml being only slightly lower. However, when higher concentrations of lipopolysaccharide were included in the emulsion particles a slightly lower uptake (Fig. 4) was obtained, possibly due to a toxic effect of the lipopolysaccharide on the leucocytes. Fig. 5 shows that the uptake by 0 5 million leucocytes during the first 6 min of incubation was essentially independent of substrate concentrations above 20 per cent (volume of opsonized emulsion added per total incubation volume). At 33 per cent of emulsion a plateau was seemingly reached indicating that at these levels of opsonized emulsion the
~I II 20 30 40 50 Lipopolysacchoride content (mg/mr )
10
sx
o
A
II /
000Ij-
~o =2
10
20 30 Emulsion (per cent)
40
Figure 5. Phagocytosis as a function of the concentration of emulsion. Normal human polymorphonuclear leucocytes (0-5 x 106) incubated for 6 min at 370 with opsonized lipopolysaccharide emulsion described in Fig. 1. The total volume of the mixture was constant (20 pI) at all concentrations of emulsion (volume of opsonized emulsion added per total incubation volume).
number and function of cells were acting as limiting factors and 33 per cent of opsonized emulsion was used in all other tests. DISCUSSION Phagocytic and bactericidal capacity of leucocytes is generally studied by the viable count technique developed by Maal0e (1946) and modified by Hirsh & Strauss (1964). The method while generally excellent is cumbersome because it involves culture
496
A. Forsgren, D. Schmeling & 0. Zettervall
of organisms and it requires a large volume of blood. A modification of the Maal0e technique has been published by Solberg (1972) which makes a separate evaluation of the phagocytic and bactericidal capacity of the cells possible. Phagocytosis of bacteria, yeast and inert particles can be studied in vitro by examination of stained smears (Hanks, 1940; Cohn & Morse, 1959; Roberts & Quastel, 1963; Brandt, 1967). Microscopic evaluation is time-consuming and difficult especially in smears containing large numbers of organisms. During the latter years radioisotope labelled particles like bentonite coated with 251I-labelled bacteria or protein (Carpenter, 1966), 32P-labelled bacteria (Trippestad & Midtvedt, 1968) or "Cr-labelled yeast cells (Nordenfelt, 1970) have been introduced for use in phagocytosis assays. The simultaneous uptake of radioactive protein from the suspending medium during phagocytosis has been the basis for another approach to obtaining quantitative measurements of phagocytosis (Chang, 1969). More recently Ward & Zvaifler (1973) introduced a simple method based on the ingestion of radiolabelled immune complexes. Two different radioactive systems are commonly used for the determination of phagocytosis. Phagocytosis of particles can be carried out with phagocytes adherent to a surface (Trippestad & Midtvedt, 1968; Nordenfelt, 1970) or the particles and phagocytes can be kept in suspension during the experimental period (Carpenter, 1966; Ward & Zvaifler, 1973). The former system has one technical advantage, the problem of separating extracellular particles from the phagocytes can possibly be solved by using cells adherent to a surface. However, it has been demonstrated by Brumfitt, Glynn & Percival (1965) that bacteria which become attached to the surface of glass-adherent cells may resist the usual mechanical methods used to remove non-phagocytosed bacteria. It is not possible to assume that glass adherent phagocytes are representative of all cells in a population as decreased glass adherence has been reported associated with a number of drugs and diseases (MacGregor, Spagnuolo & Lentnek, 1974; Penny & Galton 1966; Penny et al., 1966). In addition excessive ingestion can lead to cell detachment (Cohen & Cline, 1971). Ward & Zvaifler (1973) when using a suspension of phagocytic cells remove extracellular nonphagocytozed immune complexes by adding a large amount of antigen in order to solubilize extracellular
immune precipitate. That method has certain drawbacks. The antigen itself cannot be pal ticulate. Antibody in the form of IgG is necessary and relatively large amounts of immune complexes are needed for each test. The method cannot be used with small amounts of antigen as it requires an excess of antigen for solubilization of noningested immunoprecipitates. This excludes the possibility of using antigens which are more difficult to obtain than bovine serum albumin used by Ward & Zvaifler (1973). Neither can the method be used for quantifying serum opsonins. Oil droplets containing Oil Red 0 and coated with E. coli lipopolysaccharide are rapidly ingested by human peripheral leucocytes only if they are pretreated with serum (Stossel et al., 1972; Stossel, 1973; Stossel et al., 1973). The basis for this technique is that uningested oil drops can be separated by centrifugation from leucocytes including ingested oil. The method can be used for quantifying the opsonizing capacity of patient's sera, the influence of the alternate complement pathway on opsonization and the phagocytic function of polymorphonuclear leucocytes. It has also been used to investigate the effects of several drugs and hormones on the initial rate of phagocytosis and to approach the question of how the surface of a particle influences its acceptability as a substrate for phagocytosis. The Stossel method can also be used for isolation of intact phagocytic vesicles from human peripheral blood leucocytes (Stossel et al., 1971). The present investigation was undertaken to study whether radioactive oil particles can be used as a phagocytosis assay. The main advantages with the system developed by us are the extremely small amounts of antigen, serum and phagocytic cells required. In addition by using radioactivity a greater sensitivity can be obtained. The radioactive particles in our experiments were easily prepared and their uptake by phagocytic cells was conveniently measured without the requirement that the particles be visible or easily distinguished microscopically. Nor was there any need for the extraction of oil from the phagocytic cells as performed when using the spectrophotometric method as originally described by Stossel et al. (1972). The small amount of defined antigen required to coat the oil particles can be replaced by other antigens. It seems clear that the association of cells and radioactivity observed in these experiments is the result of phagocytosis. The suppression of uptake by
Phagocytosis of radioactive oil emulsion N-ethylmaleimide suggests that active processes in the cells were responsible for the association between cells and radioactivity. As would be expected the uptake of radioactive oil particles is related to cell concentration. The uptake was directly proportional to cell number in the range from 37,500 to 1 million polymorphonuclear leucocytes but not at higher cell numbers. Because the highest probability of detecting disturbances in cell function lies in the linear part of the curve 500,000 cells were used in the standard assay. This cell number was chosen because of the high radioactivity obtained in the cell pellet but studies of phagocytosis function could even be performed at lower cell numbers in the linear range of the curve. Under most circumstances cellular I'25 measured after 6 min of incubation adequately reflects maximum phagocytosis since further incubation results in no greater uptake. A short incubation period is preferable when dealing with phagocytes in suspension since clumping of these cells occurs rapidly at 37°. The uptake of particles by cells was significantly stimulated by normal human serum (Fig. 2) but not by heat-inactivated serum suggesting the importance of the complement system in particle opsonization. Since the radioactive content of the cell pellet is directly proportional to the concentration of serum used for opsonization the method described here is suitable for quantification of opsonins in patient's sera. As the method requires a very small amount of serum (10 p1) and neutrophils (50,000-500,000) it is very useful, especially for small children. ACKNOWLEDGMENTS We thank Dr Tor Olofsson for helpful suggestions. This work was supported by grants from the Swedish Medical Research Council. REFEREN CES BRANDT L. (1967) Studies on the phagocytic activity of neutrophilic leucocytes. Scand. J. Haematol. Supplement 2. BRuMFIrr W., GLYNN A.A. & PERCIVAL A. (1965) Factors influencing the phagocytosis of Escherichia coli. Brit. J. exp. Path. 46, 215. CARPENTER R.R. (1966) Phagocytosis by guinea pig splenic cells of Escherichia coil and protein-coated bentonite particles labelled with Iodine'25. J. Immunol. 96, 992. CHANG Y. (1969) Studies on phagocytosis. I. Uptake of radio-iodinated (131-1) human serum albumin as a
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measure of the degree of phagocytosis in vitro. Exp. cell. Res. 54, 42. COHEN A.B. & CLINE M.J. (1971) The human alveolar macrophage: Isolation, cultivation in vitro and studies of morphologic and functional characteristics. J. clin. Invest. 50, 1390. COHN L.A. & MORSE S.I. (1959) Interactions between rabbit polymorphonuclear leucocytes and staphylococci. J. exp. Med. 110, 419. COHN Z.A. (1963) The fate of bacteria within phagocytic cells. I. The degradation of isotopically labelled bacteria by polymorphonuclear leucocytes and macrophages. J. exp. Med. 117, 27. HANKS J.H. (1940) Quantitative aspects of phagocytosis as influenced by the numbers of bacteria and leucocytes. J. Immunol. 38, 159. HIRSH J.G. & STRAUSS B. (1964) Studies on heat-labile opsonin in rabbit serum. J. Immunol. 92, 145. MAAL0E 0. (1946) On the relation between alexin and opsonin. Ejnar Munksgaard, Copenhagen. MACGREGOR R.R., SPAGNUOLO P.J. & LENTNEK A.L. (1974) Inhibition of granulocyte adherence by ethanol, prednisone, and aspirin, measured with an assay system. New Engl. J. Med. 291, 642. NORDENFELT E. (1970) A method for studying phagocytosis with 51Cr-labelled yeast cells. Acta path. microbiol. scand. B, 78, 247. PENNY R., GALTON D., ScoTT J.T. & EISEN V. (1966) Studies on neutrophil function. I. Physiological and pharmacological aspects. Brit. J. Haematol. 12, 623. PENNY R. & GALTON D. (1966) Studies on neutrophil function. II Pathological aspects. Brit. J. Haematol. 12, 633. ROBERTS J. & QUASTEL J.H. (1963) Particle uptake by polymorphonuclear leucocytes and Ehrlich ascites-carcinoma cells. Biochem. J. 89, 150. SOLBERG C.O. (1972) Protection of phagocytozed bacteria against antibiotics. A new method for the evaluation of neutrophil granulocyte function. Acta med. scand. 191. 383. STOSSEL T.P., POLLARD T.D., MASON R.J. & VAUGHAN M. (1971) Isolation and properties of phagocytic vesicles from polymorphonuclear leucocytes. J. clin. Invest. 50, 1745. STOSSEL T.P., MASON R.J., HARTVIG J. & VAUGHAN M. (1972) Quantitative studies of phagocytosis by polymorphonuclear leukocytes. Use of paraffin oil emulsion to measure rate of phagocytosis. J. clin. Invest. 51, 615. STOSSEL T.P., ALPER C.A. & ROSEN F.S. (1973) Serum dependent phagocytosis of paraffin oil emulsified with bacterial lipopolysaccharide. J. exp. Med. 137, 690. STOSSEL T.P. (1973) Evaluation of opsonic and leucocyte function with a spectrophotometric test in patients with infection and with phagocytic disorders. Blood, 42, 121. TRIPPESTAD A. & MIDTVEDT T. (1968) Phagocytosis of 3Sp_ labelled E. coli by rat peritoneal polymorphonuclear leucocytes. Acta path. microbiol. scand. 74, 259. WARD P.A. & ZVAIFLER N.J. (1973) Quantitative phagocytosis by neutrophils. I. A new method with immune complexes. J. Immunol. 111, 1771. WESTPHAL 0., LUDERITZ 0. & BISTER F. (1955) Uber die Extraktion von Bakterien mit Phenol/Wasser. Zeitschrift. Naturforsh. 7B, 148.
Quantitative phagocytosis by human polymorphonuclear leucocytes USE OF RADIOLABELLED EMULSIONS TO MEASURE THE RATE OF PHAGOCYTOSIS
A. FORSGREN, D. SCHMELING* & 0. ZETTERVALL Departments of Clinical Bacteriology and Internal Medicine, University ofLund, General Hospital, Malmo, Sweden
Received 1 July 1976; acceptedfor publication 27 August 1976
opsonized by serum factors including antibodies and complement. Numerous attempts have been made to relate abnormalities in function of the neutrophil granulocytes and opsonins with the development of bacterial infection. However, difficulties in methods and lack of standardization of techniques have resulted in divergent interpretations of the experimental results. Phagocytosis can be assayed in vitro by various methods including microscopic examination of stained smears (Hanks, 1940) and methods involving cultivation of nonphagocytosed or surviving bacteria (Maal0e, 1946). Other widely used methods utilize the measurement of radioactive particles (Cohn, 1963). More recently, a phagocytosis method involving spectrophotometric determination of ingested oil has been described (Stossel, Alper & Rosen, 1973). This paper describes a method which is a modification of the spectrophotometric method described by Stossel et al. (1973) based on the quantification of ingestion of lipopolysaccharide (LPS) coated oil particles which were radioactively labelled. By this method it is possible with as little as 50,000-1 million polymorphonuclear leucocytes or 10 ,ul of serum to quantify precisely and rapidly the ingestion rate of phagocytes and the opsonic activity of serum.
Summary. A new micro-method for the quantitative measurement of phagocytosis by neutrophils is described. The material used for phagocytosis consists of a radioactive oil emulsion coated with E. coli lipopolysaccharide. Uptake of radioactive material is a function of cell number, duration of incubation, dilution of serum used for opsonization, content of lipopolysaccharide and concentration of emulsion. This method can be used to quantify rapidly and precisely phagocytosis rates of as few as 5 x 104-106 polymorphonuclear leucocytes and the opsonic activity of 10 ju1 serum.
INTRODUCTION
Phagocytosis and subsequent killing of microorganisms are major defence mechanisms against pyogenic infections. However, many bacteria are poorly ingested by phagocytic cells unless first * Present address: Department of Pediatrics, University of Minnesota, Medical School, Minneapolis, Minnesota 55455, U.S.A. Correspondence: Professor A. Forsgren, Department of Clinical Bacteriology, University of Lund, Malmb General Hospital, S-214 01 Malmo, Sweden.
491
492
A. Forsgren, D. Schmeling & 0. Zettervall
MATERIALS AND METHODS Leucocytes and sera Human peripheral leucocytes from healthy laboratory personnel were prepared by dextran sedimentation of heparinized venous blood and three times in Krebs-Ringer phosphate medium pH 7 4 and finally suspended in the same medium with the addition of 3 per cent bovine serum albumin (Poviet Producten N.V., Amsterdam) at a concentration of 5 x 107 polymorphonuclear leucocytes per ml in most experiments. Serum was collected from clotted human blood by standard laboratory techniques. The sera were stored for not more than 2-3 h at 40 or frozen immediately at -70°.
Preparation of antigen Lipopolysaccharide was extracted from Escherichia coli 0: 118 using a modification of the phenol-watermethod of Westphal, Luderitz & Bister (1955). After dialysis of the water phase against distilled water and lyophilization, the material was dissolved in a small volume of distilled water and precipitated once with ethanol in the presence of a small amount of sodium acetate. Centrifugation at 100,000 g was performed several times until free of material absorbing at 260 mp. Preparation of radioactive oil Commercial corn oil was radiolabelled with Potassium Iodide (1251) (The Radiochemical Centre, Amersham) by a chloramine T-method. One hundred pl of corn oil was dissolved in 300 p1 of chloroform. Ten pl of distilled water containing 10 ,ug of KI and 1 mCi of 25I1 followed by 40 pg of chloramine-T in 20 pul of 0 05 M phosphate buffer, pH 7 5 were added. After the development of a colour change, indicating free iodine in solution, the tube contents were mixed by shaking until the iodine colour was evenly distributed throughout the organic phase. The reaction was allowed to proceed for 1 h and then stopped by the addition of 80 pg of sodium meta bisulphite in 20 ,ul of water. Unreacted iodine was extracted from the mixture by shaking with several 5-ml portions of water. Twenty per cent of the added radioactivity was recovered in the first 5 ml of water whereafter no significant radioactive content was noted in the water. The chloroform was removed from the organic phase with a stream of nitrogen. The labelling was
performed by Mr Ingvar Larsson, The Diagnostic Isotope Laboratory, General Hospital, Malmo. Approximately 1 per cent 125I-labelled corn oil was added to, and carefully mixed with, nonradioactive diisodecylphthalate (ICN Pharmaceuticals Incorporated, Life Sciences Group, Plainview, New York) to which Red Oil 0 (Sigma Chemical Corporation) had been added (Stossel et al., 1972). Preparation of emulsions Emulsions of nonradioactive antigen and radioactive oil were prepared as follows: E. coli lipopolysaccharide was normally suspended at a concentration of 10 mg per ml in Krebs-Ringer phosphate medium in a plastic tube and dispersed by sonication for 15 seconds (MSE Ultrasonic Disintegrator, 3 mm microtip, Measuring and Scientific Equipment Ltd,
London). One volume of oil was then layered over three volumes of lipopolysaccharide suspension and the oil was emulsified into droplets by sonication according to a standardized technique. Total volume of the emulsion was usually 100,ul but occasionally a large amount of emulsion was prepared, divided into aliquots of 100 ,ul and frozen at -20°. Opsonization of the emulsion Test serum used for opsonization either undiluted or in two-fold serial dilutions in Krebs-Ringer phosphate medium containing 3 per cent albumin was incubated during rotation with an equal amount of oil emulsion at 370 for 15 min. The amount of serum used for testing of individual sera was usually 10 ,pl which was adequate for a duplicate or triplicate phagocytosis test. For other experiments larger amounts of emulsion were opsonized and divided into aliquots.
Phagocytosis procedure To siliconized round-bottomed glass tubes was added 10 p1 of a leucocyte suspension containing 101-4 x 106 polymorphonuclear leucocytes in KrebsRinger phosphate medium or in 1 MM N-ethylmaleimide (BDH Chemicals Ltd, Poole) dissolved in the same buffer. In most experiments 5 x 105 neutrophils were used. The tubes were shaken in a water bath at 370 for 5 min. Five ul of opsonized or nonopsonized emulsion was added to each tube in an experimental set in intervals of 15 s to allow for pipetting. After 5 additional min of shaking at 37° 3 ml of ice-cold 1 mM N-ethylmaleimide in 015 M
Phagocytosis of radioactive oil emulsion sodium chloride was added in 15-s intervals in the same order. The leucocytes were obtained as a pellet by centrifugation at 500 g for 10 min and the supernatant containing uningested emulsion was removed with a Pasteur-pipette and the inside of the tube was carefully wiped with a cotton swab. The cell pellet was resuspended in 3 ml of fresh saline and after centrifugation and discarding the supernatant the tube walls were wiped again and finally the 25Iradioactivity was measured in an automatic gamma counter (LKB-Wallac 80,000). All tests were done in duplicate or triplicate. RESULTS
Influence of medium on phagocytosis Table 1 shows the influence of albumin when added to a standard incubation medium used for phagocytosis experiments. Radioactivity in the cell pellet after incubation was essentially independent of the presence of albumin when an opsonized emulsion was used. However, as also indicated in the figure, an unexpectedly high radioactivity was measured in the absence of albumin when nonopsonized emulsion was tested. In some experiments an even higher radioactivity was obtained in the cell pellet when
nonopsonized emulsion was used in comparison to that recorded when the emulsion had been opsonized by serum in moderate dilutions. This shows that in the absence of albumin a cell-bound radioactivity which is nonrelated to opsonization of the oil particles can be obtained. However, as also shown in Table 1 this undesirable high uptake of nonopsonized particles can be nearly completely avoided by the addition of albumin to the incubation medium. Table 1 shows a low radioactivity in tubes without cells indicating a low degree of adherence of oil particles to the glass tube walls. However, even in the experiments without cells the radioactivity was lower when albumin was present and the radioactivity in the tubes represented 0 5 per cent of added radioactivity as compared to 1 per cent in the absence of albumin. In addition Table 1 reveals the reproducibility of the test. The coefficient of variation for values determined at 6 min in all experiments was found to be 7 per cent. Other phagocytosis experiments were performed with 0-1 per cent gelatin added to the incubation medium as previously described by Ward & Zvaifler (1973). Gelatin did not, however, significantly deter the adherence of nonopsonized particles to leucocytes. The presence of gelatin had no marked effect on phagocytosis of opsonized particles.
Table 1. Influence of albumin on cell-bound radioactivity
Neutrophils and opsonized emulsion
Albumin in medium
Radioactivity in pellet (c.p.m.)
-
2829 2858 2844 (Mean) 2590 2880 2735 (Mean) 1557 1476 1517 (Mean) 161 176 168 (Mean) 78 89 84 (Mean) 44 34 39 (Mean)
+
Neutrophils and nonopsonized emulsion
-
+ No neutrophils and nonopsonized emulsion
493
-
+
494
E
A. Forsgren, D. Schmeling & 0. Zettervall
and not only an attachment of opsonized particles to polymorphonuclear leucocytes.
800(
LO
Influence of time on phagocytosis When a constant amount of polymorphonuclear leucocytes (5 x 1O5) was incubated with a preopsonized lipopolysaccharide oil emulsion the uptake increased rapidly during the first two minutes whereafter the rate of ingestion levelled off (Fig. 2).
600( _L
z
0
400(
-c :3
n
200(
9
=
-*-i
a
,,
05 1.0 1.5 2-0 Number of polymorphonucleor leucocytes (x 106)
4.0
Figure 1. Phagocytosis as a function of cell concentration. Normal human leucocytes in a 10 p1 Krebs-Ringer phosphate medium containing 3 per cent albumin were incubated for 6 min at 370 with a constant amount of emulsion containing lipopolysaccharide (10 mg/ml) and a mixture of '251-labelled corn oil and diisodecyl phthalate. The emulsion was opsonized with undiluted serum and added to tubes in volumes of 5 pi. Each point is the mean of duplicate values. The radioactivity obtained in the cell pellet when 0-5 x 106 polymorphonuclear leucocytes were suspended in 1 mM N-ethylmaleimide is indicated by a hatched column.
c3000 -
E
i
Ci
I
2
2000-
-
1000 -
Time (min)
Influence of cell concentration on phagocytosis
Fig. 1 shows a phagocytosis experiment with 5 pl opsonized lipopolysaccharide oil emulsion and polymorphonuclear leucocytes in different numbers ranging from 10,000-4 million corresponding to 1 x 106-4 x 108 cells per ml. The uptake during the first 6 min of incubation was directly proportional to cell number in the range from 10 thousand-1 million, being 3 and 45 per cent of added emulsion respectively. A still greater uptake was obtained when higher cell numbers were used. However, at 1 x 106 cells a plateau was reached where 60 per cent of added emulsion was phagocytosed and 4 x 106 cells gave no additional uptake. The figure also indicates the influence of N-ethylmaleimide on radioactivity in the cell pellet. Cells suspended in buffer containing 1 mM N-ethylmaleimide gave a low degree of radioactivity in the cell pellet. When 5 x 105 cells (a cell number used in all other tests) were suspended in N-ethylmaleimide only 3 per cent of total added radioactivity was recovered in comparison to 23 per cent for the same number of cells suspended in buffer without N-ethylmaleimide indicating that the radioactivity of the pellet was reflecting an active phagocytio process of the cells
Figure 2. Phagocytosis as a function of time. Ten p1 of normal human leucocytes (0-5 x 106) were incubated with 5 jul of the opsonized emulsion described in Fig. I (closed circles). For comparison the rate of phagocytosis of a nonopsonized emulsion is included in the figure (open circles).
After 6 min a plateau was reached with an uptake of 23 per cent of added radioactivity and no further uptake was obtained. Uptake of nonopsonized particles was strikingly lower than that with opsonized particles. A plateau was also reached with nonopsonized particles the level of this being 11 per cent of the values obtained with opsonins present. Influence of opsonization on phagocytosis Fig. 3 reveals that the opsonic activity of human serum as determined in the standard assay system was limiting and was proportional to serum concentration in the range of 12 5-100 per cent corresponding to serum dilutions 1:8 and undiluted. The percentage uptake of added radioactivity was 23, 15, 1 1, and 8 for serum dilutions undiluted 1: 2, 1:4, and 1: 8, respectively. When lower concentrations of serum were used for opsonization a sharply decreasing rate of phagocytosis was obtained. At a serum
495
Phagocytosis of radioactive oil emulsion tD
E w
3000
{
E
E >
3000
2000
_
2000
-0
1000 _
1000
/
0
0
12!5 25
50
100
Serum (per cent)
Figure 3. Phagocytosis as a function of opsonization. Ten microlitres of normal human leucocytes (5 x 107 per ml) were incubated with a lipopolysaccharide-oil emulsion preopsonized with varying concentrations of serum in a final volume of 5 j1u. Nonopsonized emulsion was included as a control. The radioactivity obtained when emulsion was opsonized with heated serum is indicated by a hatched column.
Figure 4. Phagocytosis as a function of content of lipopolysaccharide in suspension used for preparation of emulsion. Normal human leucocytes in 10 p1 medium incubated for 6 min at 370 with 5 p1 of emulsified oil coated with varying amounts of lipopolysaccharide. Each point is the mean of duplicate values.
(0
200C Id
concentration of approximately 3 per cent (serum dilution: 1:32) no appreciable difference was obtained compared to the control where no serum was used. Fig. 3 also shows the low degree of radioactivity obtained in the cell pellet when an emulsion opsonized with heat-inactivated serum (560, 30 min) was used. Influence of emulsion content and concentration on phagocytosis As shown in Fig. 4 maximal uptake was obtained when E. coli lipopolysaccharide in a concentration of 10 mg per ml was used for preparation of the emulsion, the uptake at 5 mg per ml being only slightly lower. However, when higher concentrations of lipopolysaccharide were included in the emulsion particles a slightly lower uptake (Fig. 4) was obtained, possibly due to a toxic effect of the lipopolysaccharide on the leucocytes. Fig. 5 shows that the uptake by 0 5 million leucocytes during the first 6 min of incubation was essentially independent of substrate concentrations above 20 per cent (volume of opsonized emulsion added per total incubation volume). At 33 per cent of emulsion a plateau was seemingly reached indicating that at these levels of opsonized emulsion the
~I II 20 30 40 50 Lipopolysacchoride content (mg/mr )
10
sx
o
A
II /
000Ij-
~o =2
10
20 30 Emulsion (per cent)
40
Figure 5. Phagocytosis as a function of the concentration of emulsion. Normal human polymorphonuclear leucocytes (0-5 x 106) incubated for 6 min at 370 with opsonized lipopolysaccharide emulsion described in Fig. 1. The total volume of the mixture was constant (20 pI) at all concentrations of emulsion (volume of opsonized emulsion added per total incubation volume).
number and function of cells were acting as limiting factors and 33 per cent of opsonized emulsion was used in all other tests. DISCUSSION Phagocytic and bactericidal capacity of leucocytes is generally studied by the viable count technique developed by Maal0e (1946) and modified by Hirsh & Strauss (1964). The method while generally excellent is cumbersome because it involves culture
496
A. Forsgren, D. Schmeling & 0. Zettervall
of organisms and it requires a large volume of blood. A modification of the Maal0e technique has been published by Solberg (1972) which makes a separate evaluation of the phagocytic and bactericidal capacity of the cells possible. Phagocytosis of bacteria, yeast and inert particles can be studied in vitro by examination of stained smears (Hanks, 1940; Cohn & Morse, 1959; Roberts & Quastel, 1963; Brandt, 1967). Microscopic evaluation is time-consuming and difficult especially in smears containing large numbers of organisms. During the latter years radioisotope labelled particles like bentonite coated with 251I-labelled bacteria or protein (Carpenter, 1966), 32P-labelled bacteria (Trippestad & Midtvedt, 1968) or "Cr-labelled yeast cells (Nordenfelt, 1970) have been introduced for use in phagocytosis assays. The simultaneous uptake of radioactive protein from the suspending medium during phagocytosis has been the basis for another approach to obtaining quantitative measurements of phagocytosis (Chang, 1969). More recently Ward & Zvaifler (1973) introduced a simple method based on the ingestion of radiolabelled immune complexes. Two different radioactive systems are commonly used for the determination of phagocytosis. Phagocytosis of particles can be carried out with phagocytes adherent to a surface (Trippestad & Midtvedt, 1968; Nordenfelt, 1970) or the particles and phagocytes can be kept in suspension during the experimental period (Carpenter, 1966; Ward & Zvaifler, 1973). The former system has one technical advantage, the problem of separating extracellular particles from the phagocytes can possibly be solved by using cells adherent to a surface. However, it has been demonstrated by Brumfitt, Glynn & Percival (1965) that bacteria which become attached to the surface of glass-adherent cells may resist the usual mechanical methods used to remove non-phagocytosed bacteria. It is not possible to assume that glass adherent phagocytes are representative of all cells in a population as decreased glass adherence has been reported associated with a number of drugs and diseases (MacGregor, Spagnuolo & Lentnek, 1974; Penny & Galton 1966; Penny et al., 1966). In addition excessive ingestion can lead to cell detachment (Cohen & Cline, 1971). Ward & Zvaifler (1973) when using a suspension of phagocytic cells remove extracellular nonphagocytozed immune complexes by adding a large amount of antigen in order to solubilize extracellular
immune precipitate. That method has certain drawbacks. The antigen itself cannot be pal ticulate. Antibody in the form of IgG is necessary and relatively large amounts of immune complexes are needed for each test. The method cannot be used with small amounts of antigen as it requires an excess of antigen for solubilization of noningested immunoprecipitates. This excludes the possibility of using antigens which are more difficult to obtain than bovine serum albumin used by Ward & Zvaifler (1973). Neither can the method be used for quantifying serum opsonins. Oil droplets containing Oil Red 0 and coated with E. coli lipopolysaccharide are rapidly ingested by human peripheral leucocytes only if they are pretreated with serum (Stossel et al., 1972; Stossel, 1973; Stossel et al., 1973). The basis for this technique is that uningested oil drops can be separated by centrifugation from leucocytes including ingested oil. The method can be used for quantifying the opsonizing capacity of patient's sera, the influence of the alternate complement pathway on opsonization and the phagocytic function of polymorphonuclear leucocytes. It has also been used to investigate the effects of several drugs and hormones on the initial rate of phagocytosis and to approach the question of how the surface of a particle influences its acceptability as a substrate for phagocytosis. The Stossel method can also be used for isolation of intact phagocytic vesicles from human peripheral blood leucocytes (Stossel et al., 1971). The present investigation was undertaken to study whether radioactive oil particles can be used as a phagocytosis assay. The main advantages with the system developed by us are the extremely small amounts of antigen, serum and phagocytic cells required. In addition by using radioactivity a greater sensitivity can be obtained. The radioactive particles in our experiments were easily prepared and their uptake by phagocytic cells was conveniently measured without the requirement that the particles be visible or easily distinguished microscopically. Nor was there any need for the extraction of oil from the phagocytic cells as performed when using the spectrophotometric method as originally described by Stossel et al. (1972). The small amount of defined antigen required to coat the oil particles can be replaced by other antigens. It seems clear that the association of cells and radioactivity observed in these experiments is the result of phagocytosis. The suppression of uptake by
Phagocytosis of radioactive oil emulsion N-ethylmaleimide suggests that active processes in the cells were responsible for the association between cells and radioactivity. As would be expected the uptake of radioactive oil particles is related to cell concentration. The uptake was directly proportional to cell number in the range from 37,500 to 1 million polymorphonuclear leucocytes but not at higher cell numbers. Because the highest probability of detecting disturbances in cell function lies in the linear part of the curve 500,000 cells were used in the standard assay. This cell number was chosen because of the high radioactivity obtained in the cell pellet but studies of phagocytosis function could even be performed at lower cell numbers in the linear range of the curve. Under most circumstances cellular I'25 measured after 6 min of incubation adequately reflects maximum phagocytosis since further incubation results in no greater uptake. A short incubation period is preferable when dealing with phagocytes in suspension since clumping of these cells occurs rapidly at 37°. The uptake of particles by cells was significantly stimulated by normal human serum (Fig. 2) but not by heat-inactivated serum suggesting the importance of the complement system in particle opsonization. Since the radioactive content of the cell pellet is directly proportional to the concentration of serum used for opsonization the method described here is suitable for quantification of opsonins in patient's sera. As the method requires a very small amount of serum (10 p1) and neutrophils (50,000-500,000) it is very useful, especially for small children. ACKNOWLEDGMENTS We thank Dr Tor Olofsson for helpful suggestions. This work was supported by grants from the Swedish Medical Research Council. REFEREN CES BRANDT L. (1967) Studies on the phagocytic activity of neutrophilic leucocytes. Scand. J. Haematol. Supplement 2. BRuMFIrr W., GLYNN A.A. & PERCIVAL A. (1965) Factors influencing the phagocytosis of Escherichia coli. Brit. J. exp. Path. 46, 215. CARPENTER R.R. (1966) Phagocytosis by guinea pig splenic cells of Escherichia coil and protein-coated bentonite particles labelled with Iodine'25. J. Immunol. 96, 992. CHANG Y. (1969) Studies on phagocytosis. I. Uptake of radio-iodinated (131-1) human serum albumin as a
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measure of the degree of phagocytosis in vitro. Exp. cell. Res. 54, 42. COHEN A.B. & CLINE M.J. (1971) The human alveolar macrophage: Isolation, cultivation in vitro and studies of morphologic and functional characteristics. J. clin. Invest. 50, 1390. COHN L.A. & MORSE S.I. (1959) Interactions between rabbit polymorphonuclear leucocytes and staphylococci. J. exp. Med. 110, 419. COHN Z.A. (1963) The fate of bacteria within phagocytic cells. I. The degradation of isotopically labelled bacteria by polymorphonuclear leucocytes and macrophages. J. exp. Med. 117, 27. HANKS J.H. (1940) Quantitative aspects of phagocytosis as influenced by the numbers of bacteria and leucocytes. J. Immunol. 38, 159. HIRSH J.G. & STRAUSS B. (1964) Studies on heat-labile opsonin in rabbit serum. J. Immunol. 92, 145. MAAL0E 0. (1946) On the relation between alexin and opsonin. Ejnar Munksgaard, Copenhagen. MACGREGOR R.R., SPAGNUOLO P.J. & LENTNEK A.L. (1974) Inhibition of granulocyte adherence by ethanol, prednisone, and aspirin, measured with an assay system. New Engl. J. Med. 291, 642. NORDENFELT E. (1970) A method for studying phagocytosis with 51Cr-labelled yeast cells. Acta path. microbiol. scand. B, 78, 247. PENNY R., GALTON D., ScoTT J.T. & EISEN V. (1966) Studies on neutrophil function. I. Physiological and pharmacological aspects. Brit. J. Haematol. 12, 623. PENNY R. & GALTON D. (1966) Studies on neutrophil function. II Pathological aspects. Brit. J. Haematol. 12, 633. ROBERTS J. & QUASTEL J.H. (1963) Particle uptake by polymorphonuclear leucocytes and Ehrlich ascites-carcinoma cells. Biochem. J. 89, 150. SOLBERG C.O. (1972) Protection of phagocytozed bacteria against antibiotics. A new method for the evaluation of neutrophil granulocyte function. Acta med. scand. 191. 383. STOSSEL T.P., POLLARD T.D., MASON R.J. & VAUGHAN M. (1971) Isolation and properties of phagocytic vesicles from polymorphonuclear leucocytes. J. clin. Invest. 50, 1745. STOSSEL T.P., MASON R.J., HARTVIG J. & VAUGHAN M. (1972) Quantitative studies of phagocytosis by polymorphonuclear leukocytes. Use of paraffin oil emulsion to measure rate of phagocytosis. J. clin. Invest. 51, 615. STOSSEL T.P., ALPER C.A. & ROSEN F.S. (1973) Serum dependent phagocytosis of paraffin oil emulsified with bacterial lipopolysaccharide. J. exp. Med. 137, 690. STOSSEL T.P. (1973) Evaluation of opsonic and leucocyte function with a spectrophotometric test in patients with infection and with phagocytic disorders. Blood, 42, 121. TRIPPESTAD A. & MIDTVEDT T. (1968) Phagocytosis of 3Sp_ labelled E. coli by rat peritoneal polymorphonuclear leucocytes. Acta path. microbiol. scand. 74, 259. WARD P.A. & ZVAIFLER N.J. (1973) Quantitative phagocytosis by neutrophils. I. A new method with immune complexes. J. Immunol. 111, 1771. WESTPHAL 0., LUDERITZ 0. & BISTER F. (1955) Uber die Extraktion von Bakterien mit Phenol/Wasser. Zeitschrift. Naturforsh. 7B, 148.
