Journal of Immunological Methods, 8 (1975) 213--222 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
QUANTITATIVE ASSAYS OF HUMAN MONOCYTE-MACROPHAGE FUNCTION
WILLIAM L. WESTON, ROBERTA D. DUSTIN and STEVEN K. HECHT
Division of Dermatology, Department of Medicine, University of Colorado School of Medicine, Denver, Colorado, U.S.A. (Received 17 December 1974, accepted 17 February 1975)
Monocyte-macrophages are required for the development of cell mediated immunity to a variety of microorganisms and tumors. Quantitative assays of human monocytemacrophage function would be most useful in the evaluation of cell mediated immune function in man. Five quantitative assays are described that provide a human monocytemacrophage function profile. These assays parallel the physiologic steps necessary for monocyte-macrophages to function as phagocytes: 1) chemotaxis, 2) opsonization, 3) phagocytosis, 4) phagocytosis-induced metabolic stimulation and 5) destruction of foreign material. Application of these quantitative assays will allow detection and dissection of disorders of monocyte-macrophage function in man.
INTRODUCTION
Quantitative assays of human monocyte-macrophage function should take into account the physiologic mechanisms necessary for these cells to perform as phagocytes. These monocyte-macrophage functions can be divided into a series of physiologic steps, each of which can be quantitated: 1) cell movement, 2) adherence of particles to this cell surface, 3) engulfment (phagocytosis), 4) stimulation of intracellular metabolism and 5) destruction of foreign material. Five quantitative assays of human monocyte-macrophage function have been performed to measure these steps: 1) chemotaxis, 2) opsonization of the yeast particles, 3) phagocytosis of (a) paraffin oil and (b) bacteria, 4) phagocytosis-induced Nitro Blue Tetrazolium (NBT) reduction and 5) bactericidal assay for Staphylococcus aureus. This allows compilation of a monocyte-macrophage function profile in evaluating human host defenses. Stimulation or activation of monocyte-macrophages is a common, perhaps invariable, requirement for the development of cell mediated immunity (CMI) against both tumors and microorganisms (Bjorklund et al., 1972; Supported in part by NIH Research Grant, #AM 16752 Reprint requests to be sent to William L. Weston, Division of Dermatology, University of Colorado Medical Center, 4200 East Ninth Avenue, Denver, Colorado 80220.
214
Lejeune and Evans, 1972; Lohmann-Matthes, 1972; Evans, 1973; Nelson, 1974). There is an increasing awareness of the importance of the monocytemacrophage in host defense against tumors and microorganisms (Evans, 1973; Nelson, 1974). Quantitative assays of monocyte-macrophage function are essential for the evaluation of host defenses in a great variety of clinical settings. MATERIALS AND METHODS
Separation of rnonocytes Ten to 15 ml of heparinized (sodium heparin, 10 units/ml) venous blood was withdrawn from 20 healthy young adult donors, ages 25--35. The blood was divided into aliquots of 4 ml, each of which was layered over 1.5 ml of 9% Ficoll-Hypaque (sodium diatrizole) solution in 6-ml plastic test tubes after the technique of B o y u m (1968). The Ficoll-Hypaque solution contains 10 parts of Hypaque to 24 parts of Ficoll. After centrifugation at 400 g for 40 min a distinct layer of mononuclear leukocytes appeared which was removed carefully with a Pasteur pipette and transferred to a 12-ml plastic test tube. The cell suspension was washed three times with RPMI-1640 tissue culture media (Grand Island Biological Company, Grand Island, New York) and centrifuged at 200 g for 10 min. The cells were resuspended in 1 ml of RPMI-1640. Further purification of the monocytes was obtained by layering the cell pellet over a 28% bovine serum albumin (Path-O-Cyte 5, Miles Laboratories, Elkhart, Indiana) gradient and centrifuging at 400 g for 50 rain. The m o n o c y t e layer on t o p was aspirated and cell numbers were counted with a Unopette dilution system and a standard hemocytometer. Trypan Blue viability (Waithe and Hirschhorn, 1973) and neutral red phagocytosis (Gesner and Howard, 1967) were performed on the cells to determine the percentage of phagocytotic cells and their viability. All assays were adjusted for the percentage of phagocytic mononuclear cells in the final preparation.
Chemotaxis assay Chemotaxis was modified from the method of Boyden (1962). Stainless steel chambers of the Sykes--Moore variety were utilized and separated into two compartments by two Millipore filters. We used an 8.0-micron upper filter and 0.45-micron lower filter allowing measurement of any cells that may pass through the upper filter due to adherence defects and be entrapped on the lower Millipore filter (Keller et al., 1972; Frei et al., 1974). The standard chemotactic attractant used in our assay is a 10% solution of human serum in RPMI-1640 tissue culture media activated with E. coli lipopolysaccharide endotoxin type O l 1 1 : B 4 (Difco Laboratories, Detroit, Michigan) at 200 pg/ml. (Ward, 1968; Baum, 1971). This amount of endotoxin has been found to be the most effective in activating the C3 and C5
215 components of the complement system (Gewurz, 1968). All serum used in the assay was obtained from a single donor and used throughout the study giving a standard attractant. 0.5-ml aliquots of the serum were stored and frozen at --70°C. Attractants were added by injection into the lower chamber of the Sykes--Moore chamber. 1 X 106 m o n o c y t e s / m l were added to the upper c o m p a r t m e n t and all assays were performed in triplicate. Chambers were incubated at 37°C with 5% CO2 and 100% h u m i d i t y for 5 hr. After incubation the filters were removed and fixed in absolute methanol and rinsed in distilled water. Filters were stained using hematoxylin and blueing agents and then dehydrated in a standard fashion and cleared with xylene. Filters were dried overnight and then trimmed and m o u n t e d on glass slides with cover slips. Ten high power fields were counted. The cells on the upper surface of the 8.0-micron filter (cells that did not migrate) were compared to those migrating through the filter (including those within the filter) plus those that passed through the 8.0-micron filter and were entrapped on the surface of the 0.45-micron filter. The results were expressed as the percentage of total cells migrating by the formula: Total number of cells migrating Total number of cells n o t migrating
X 100
By expressing the results as the percentage of mononuclear leukocytes migrating, a means of comparison between investigators using the same procedures is provided and a precise number of cells does not have to be delivered to the upper membrane surface.
Monocy te phagocy tosis and phagocy tosis-induced NBT reduction The m e t h o d of Stossel (Stossel et al., 1972; Stossel, 1973) for simultaneous measurement of phagocytosis and NBT reduction by phagocytic cells was adapted for monocytes. This m e t h o d of phagocytosis was chosen because it avoids the adherence of phagocytic material to cell surfaces giving a false appearance of internalization of particles. Paraffin oil (Fisher Chemical Company, Fairlawn, New Jersey) does not adhere to cell surfaces (Stossel, 1973). The m o n o c y t e concentration was adjusted to 1 X 106 monocytes/ml. 500 mg of Oil Red O (Paragon C and C Company, Bronx, New York) was placed in a flask containing 0.5 ml of chloroform. This mixture was diluted to 20 ml with paraffin oil at a density of 0.89. These substances were mixed overnight and centrifuged at 2 0 0 g for 1 hr. The supernatant fluid was pooled in an Erlenmeyer flask and chloroform removed by evaporation overnight at 50°C under vacuum. It was again centrifuged at 2000 g for 1 hr. The optical density of the Oil Red O solution at 525 nanometers was used to compute a constant (K) for converting optical density of mg of paraffin oil ingested where:
216 1/O.D. K - - × 0.89 ml of dye Optical density of the final solution was found to undergo no change over a period of weeks (Stossel, 1973). The emulsion was prepared with 30 mg of E. coli lipopolysaccharide O l l l : B4 prepared by the Bouin procedure (Difco Laboratories, Detroit, Michigan). This was suspended in 3 ml of Hank's balanced salt solution (Difco Laboratories, Detroit, Michigan} in a 6-ml plastic test tube. The lipopolysaccharide was dispersed by vortex mixing for 15 seconds, then 1 ml of paraffin off containing Oil Red O was layered over the lipopolysaccharide suspension. The oil was emulsified into droplets by vortex mixing for 60 sec. Aliquots of this emulsion were frozen at --20°C. NBT was suspended in Hank's balanced salt solution at a concentration of 2 mg/ml and filtered through a 0.9.2 Millipore filter. Standardization was accomplished by adding 0.1 ml of the filtrate to 0.04 ml of 1 mM ascorbic acid and 0.2 normal sodium hydroxide. This reduced the NBT to formazan and the entire filter was dissolved i n 2 ml of p-dioxane with heating at 85°C and the optical density was determined immediately at 580 nm on a spectrophotometer. The conversion factor from the optical density of pg of formazan has been established by previous investigators as 14.14 tLg of formazan per optical density unit for 1 cm light path in dioxane at 25°C (Stossel, 1973). Serum, either used fresh or frozen at --70°C, was divided into 0.4 ml aliquots and incubated with 0.4 ml of the paraffin oil particles at 37°C for 15 min to opsonize the emulsion. A simultaneous assay was performed by adding 0.015 ml of 0.2 M potassium cyanide in Hank's balanced salt solution in two separate Falcon plastic test tubes that contained 0.4 ml of the leukocyte suspension. 0.4 ml of Hank's balanced salt solution was added to the first tube and 0.4 ml of the NBT solution was added to the second. After shaking in a water bath of 37°C for 5 min, 0.2 ml of emulsified Oil Red O particles opsonized by patients serum was added and the mixture incubated for 5 min at 37°C in the water bath. Six ml of ice cold 1 mM N-ethylmaleimide in 0.15 M sodium chloride was added and the uningested emulsion removed by centrifugation at 500 g for 15 min. The supernatant was discarded and the leukocyte pellet resuspended by adding 6 ml of fresh 0.15 M sodium chloride. The mixture was centrifuged at 5 0 0 g and the supematant discarded. Oil Red O and formazan were extracted from the pellets by adding 3 ml of p-dioxane and heating the extracts at 85°C for 15 min. Optical density solutions were clarified by centrifugation at 1000 g for 15 min and then determined at 525 nm and 580 nm. Results were expressed as mg of paraffin oil ingested per min per 107 monocytes. Results were calculated for the following formula: O.D.s2s X 50 Leukocytes/ml × m o n o c y t e s / 1 0 0 leukocytes
XK
217 NBT was expressed as pgm of formazan generated per min per 107 monocytes as derived from the following formula: O.D.ss0 in the extract with formazan -- O.D.ss0 in the extract w i t h o u t formazan X 50 X 14.14 Leukocytes/ml X monocytes/100 leukocytes
Monocyte attachment o f opsonized yeast Opsonization of yeast by the method of Miller (1969) was modified for monocytes. Preservative free bakers yeast was prepared by dissolving 100 ml of yeast in 8 ml of 0.9% NaC1 and incubating in a boiling water bath for 30 min to kill the yeast. The yeast was then filtered and the concentration was adjusted to 20 X 106 yeast particles/ml in a solution of 0.9% sodium chloride. 0.5 X 10 ~ monocytes/ml were mixed with 0.5 ml of AB negative h u m a n serum as an opsonin in a 6 ml plastic test tube. 0.05 ml of the 20 X 10 ~ yeast particle suspension was added to the mixture yielding a yeast : m o n o c y t e ratio of 1 : 5. The mixture was incubated at 37°C with 5% CO2 and 100% h u m i d i t y and aliquots removed after 5, 15 and 60 min of incubation with constant tilting and mixing. Thin layer smears were prepared of the aliquots and Wright's stained slides were counted and the number of yeast particles ingested/cell was determined as well as the percent of cells ingesting or attaching yeast.
Monocyte bactericidal assay and bacterial phagocytosis By utilizing the m e t h o d modified from Tan et al., (1971) it is possible to separate killing from phagocytosis. This is accomplished by the use of lysostaphin, a p o t e n t anti-staphylococcal agent that does not enter the cell. By destroying all the extra-cellular staphylococci one can measure only those organisms that have been engulfed. Defects in phagocytosis would otherwise masquerade as defects in killing because viable bacteria would remain on the cell surface. An inoculum of Staphylococcus aureus 502A was cultured overnight in trypticase soy broth. The bacteria was centrifuged and the pellet resuspended in 0.9% sodium chloride and centrifuged again at 50g. The bacterial pellet was diluted with Hank's balanced salt solution until the optical density at 550 nm was 0.3. This optical density corresponded to approx. 10 X 106 viable bacteria/ml. 10 X 106 monocytes in 1 ml were transferred with a siliconized Pasteur pipette and centrifuged at 200 g for 5 min. The supernatant was discarded and the pellet resuspended in 0.05 ml of sodium heparin and 0.5 ml of 0.1% Hank's gel (0.1% gelatin USP, J.T. Baker Company, in Hank's balanced salt solution, sterilized by autoclaving), with a siliconized sterile Pasteur pipette. One ml of AB negative human pooled serum was diluted with 4 ml of Hank's balanced salt solution to make a 20% opsonization solution. The solution was mixed for 5 min and 0.5 ml
218 o f this solution was added to 0.5 ml of the m o n o c y t e suspension. In a control tube m o n o c y t e s were o m i t t e d and 0.5 ml of Hank's balanced salt solution was added. 0.1 ml of t he bacterial suspension was added to each tube. The tubes were mixed gently and a 0.1 ml aliquot was removed and diluted in 10 ml of sterile distilled water. These were mixed for 30 sec and a fu rth er 1 to 100 dilution was made by diluting 0.1 ml of 10 ml distilled water. These were mixed by inverting and a 0.1 ml aliquot was pipetted and streaked over a trypticase soy agar Petri dish for a baseline (O) col ony count. All plates were assayed in duplicate. The tubes were then tightly capped and placed on a tilting table at 37°C incubation with 100% humidity for 2 hr. 0.2 ml o f each sample was transferred to a 6-ml plastic tube and 1 ml of filtered lysostaphin. 500 units/ml were added to each tube and placed in a 37°C water bath for 20 min. 0.1 ml was pipetted from each of the original mixtures o f cells and staphylococcal organisms and diluted in 10 ml of distilled water. These tubes were mixed for 30 seconds and a 0.1 ml aliquot was removed and rediluted in 10 ml of distilled water. In the patient test samples, 0.1 ml of this dilution was diluted in 2 ml of distilled water. The tubes were again mixed by inverting and 0.1 ml o f each tube was pipetted into trypticase soy agar plates, streaked and incubated at 37°C overnight. Similar dilutions were made for the lysostaphin after neutralization with 0.1 ml o f 2.5% trypsin and then incubated overnight in the bacterial incubator at 37°C. All plates were c o u n t e d using a view box after the overnight incubation. A percent of total viable bacteria (%TVB) was calculated by the following formula: %TVB =
No. colonies at 2 hr (2.1 X 104) No. colonies at 0 hr (1 X l 0 s)
The p er cen t o f intracellular bacteria (%IB) was calculated from the following formula: No. colonies at 2 hr after lysostaphin (1.05 X 104) %IB
=
No. colonies at 0 hr (1 X 105)
The p er cen t o f extracellular bacteria retaining (%EB) was calculated by the following formula: %EB
= %TVB
-- %IB
RESULTS The results are designed as a m o n o c y t e - m a c r o p h a g e funct i on profile and are expressed as the mean -+2 standard deviations as shown in the table. Chemotaxis is best expressed as the percent of total cells migrating as a standard frame of reference for o t h e r m e t ho ds of chemotaxis as suggested by
219 TABLE 1 Monocyte-macrophage function profile. Assay
Mean ± 2 S.D.
Chemotaxis (% cells responding)
11.62 +- 6.04
Opsonization (% cells with yeast adhering)
5 min 20.4 + 11.1 15 min 38 ± 21.6 60 min 43.7 + 38
Phagocytosis (pg paraffin oil ingested/min/107 monocytes) (% extracellular bacteria surviving)
0.153 ± 0.072 17.7
Intracellular metabolism (pg formazan reduced/minute/107 monocytes) 391 Killing (30 controls) (% intracellular bacteria surviving)
± 10.6 -+ 203
0.77 -+ 0.40
Naidu and Neubould (1974). A t t a c h m e n t appeared to show optimal differences after 15 min of exposure of opsonized yeast particles. At 60 min the variability was t o o great to permit distinction bet w een normal and abnormal states. The percent of total viable bacteria and the bactericidal assay is a useful screening procedure for bot h phagocytosis and the bactericidal assay. By calculating the pe r cent of intracellular bacteria surviving an accurate measure of those organisms phagocytized and killed is obtained. Defects in phagocytosis would otherwise mask these results and result in a faulty d e t e r m i n a t i o n o f defective intracellular killing o f bacteria (Tan et al., 1971; Van F u r th and Van Zwet, 1973). DISCUSSION Despite the i m p o r t a n c e of m onocyt e- m acrophages in host defenses against infections and t um or s there has been a paucity of reports on m o n o c y t e macrophage f u n c t i o n in humans. Some investigation of m o n o c y t e function in humans has been directed toward in vitro chemotaxis (Ward, 1968; Snyderm an et al., 1972) and in vivo cell m o v e m e n t (Meuret and H of f m a n, 1973). Defective m o n o c y t e chemotaxis has been det ect ed in patients with cancer (Snyderm an et al., 1974; S n y d e r m a n and Stahl, 1974) and a child with chronic moniliasis (Snyderman, 1973). M o n o c y t e phagocytic f u n c t i o n f or IgG-coated e r y t h r o c y t e s (Huber, 1968) has been investigated and shown to be diminished by Rifampin (Urbanitz, 1974). Phagocytosis of bacteria has been f o u n d to be normal in children
220 with neutropenia and chronic infections (Baehner and Johnson, 1972) and in m y e l o m o n o c y t i c leukemia (Cline, 1973) and lymphoma. Monocyte bactericidal activity was normal in children with neutropenia (Baehner and Johnson, 1972) but defective in leukemia and l y m p h o m a (Cline, 1973). A candicidal assay for m o n o c y t e s has been developed as well (Lehrer and Cline, 1969). Simultaneous m o n o c y t e functions have rarely been reported in humans (Baehner and Johnson, 1972; Cline, 1973) and then only for phagocytosis and killing b u t n o t for cell movement. More attention has recently been focused on the monocyte-macrophage and its role in host defense. Monocytes synthesize a large number of biologically important products including transferrin, complement, interferon, pyrogens and granulopoietin (Lobuglio, 1973). It has recently been postulated that impaired activity of the m o n o c y t e may explain a number of unusual infectious diseases seen in the newborn (Gotoff, 1974). The importance of the macrophage in clinical disease states (Cline, 1973; Meuret and Hoffmann, 1973; Snyderman, 1973; Snyderman et al., 1974; Urbanitz, 1974) and in t u m o r immunology (Evans, 1973; Nelson, 1974) from early studies suggests we are at the threshold of understanding the role of the macrophage in host defenses. The quantitative monocyte-macrophage assays described here offer several advantages in investigation of the m o n o c y t e ' s role in host defense. First, relatively small amounts of blood are needed to obtain this profile provided the subjects studied have normal peripheral white counts and often 7--10 ml of blood will suffice in children with elevated white counts whereas leukopenic patients may require large volumes of blood (30--40 ml). Further microquantitation of these assays will be required to facilitate investigation of leukopenic subjects. Second, preliminary cost analysis revealed that this complete profile costs $30.00 per subject excluding technician salaries. Laboratory equipment needed includes a centrifuge, bacterial incubator and tissue culture incubator in addition to the usual laboratory equipment (microscope, balance, etc.). An available autoclave also reduces costs. Thirdly, a maximum of 4 profiles can be performed in a day. It takes approx. 2 hr to separate blood and an average of 4 hr for most assays. Assays can be completed within an 8-hr working day. It would appear that 3 different assays of phagocytosis are included in the monocyte-macrophage function profile. However, only one of these assays measures phagocytosis; the others are included to measure other functions that are closely related to phagocytosis. Phagocytosis of bacteria must be included in the bactericidal assay so that one can distinguish between faulty killing and faulty phagocytosis within that particular assay. If bacteria were not ingested they could not be destroyed, resulting in a high percentage of viable bacteria remaining leading to the false conclusion that it was measuring faulty killing. The most accurate measurement of phagocytosis is that of
221
paraffin oil ingestion since paraffin oil does not adhere to cell surfaces and all measured is that ingested. The third assay, utilizing opsonized yeast, really measures attachment of yeast since there is no method to distinguish between the yeast on the m o n o c y t e surface and that ingested. It is incorrect to consider this assay a phagocytic assay and it is included as an assay to measure attachment to the m o n o c y t e surface. Substituting another assay such as surface receptors for IgG or a cytotoxicity assay may be desired by other investigators. Development of these quantitative assays of human monocyte-macrophage function which take into account the physiologic mechanisms of these cells acting as phagocytes will provide the data to define the role of the monocyte-macrophage in host defenses.
REFERENCES Baehner, R.L., and R.B. Johnson, Jr., 1972, Blood 40, 31. Baum, J., 1971, J. Lab. Clin. Med. 77,501. Bennett, W.E., and Z.A. Cohn, 1966, J. Exp. Med. 123, 145. Bjorklund, B., V. Bjorklund, R. Lundstrom, G. Eklund, L. Nilsson, R. Gronneberg, 1972, J. Reticuloendothel. Soc. 11, 29. Boyden, S.V., 1962, J. Exp. Med. 115, 453. Boyum, A., 1968, Scand. J. Clin. Lab. Invest. 21, I {Suppl. 97). Cline, M.J., 1973, J. Clin. Invest. 52, 2185. Evans, R., 1973, Brit. J. Cancer 28, 19. Frei, P.C., M.H. Baisero, M. Ochsner, 1974, J. Immunol. Methods 5,355. Gesner, B.M., and J.G. Howard, 1967, in: Handbook of experimental immunology, ed., D.M. Weir (F.A. Davis Company, Philadelphia, Pennsylvania) p. 1009. Gewurz, H., 1968, J. Exp. Med. 128, 149: Gotoff, S.P., 1974, J. Pediatrics 85, 149. Huber, H., 1968, Int. Arch. Allergy 34, 18. Keller, H.U., J.F. Borel, D.C. Wilkinson, M.W. Hess, H. Collier, 1972, J. Immunol. Methods 1, 165. Lehrer, R.I., and M.J. Cline, 1969, J. Bacteriol. 98, 996. Lejeune, F., and R. Evans, 1972, Eur. J. Cancer 8, 549. Lobuglio, A.Z., 1973, New Eng. J. Med. 288,212. Lohmann-Metthes, M.L., H. Schipper, H. Fischer, 1972, Eur. J. Immunol. 2, 45. Meuret, G., and G. Hoffmann, 1973, Brit. J. Haematol. 24, 225. Miller, M.E., 1969, J. Pediatrics 74, 255. Naidu, T.G., and G.H.S. Neubould, 1974, Immunol. Communications 3, 457. Nelson, D.S., 1974, Transplant Rev. 19, 226. Snyderman, R., L.C. Altman, M.J. Hausman, S.E. Mergenhagen, 1972, J. Immunol. 108, 857. Snyderman, R., 1973, Ann. Int. Med. 78,509. Snyderman, R., J. Dickson, L. Meadows, M. Pike, 1974, Clin. Res. 22, 430A {Abstract). Snyderman, R., and C. Stahl, 1975, in: The phagocytic cell in host resistance, eds. J.A. Bellanti and D.H. Dayton {Raven Press, New York, N.Y.). Stossel, T.P., R.J. Mason, J. Harting, 1972, J. Clin. Invest, 51,615. Stossel, T.P., 1973, Blood, 42, 121. Stossel, T.P., 1974, New Engl. J. Med. 290, 717,774, and 833 (three parts).
222 Tan, J.S., C. Watanakunakorn, J.P. Phair, 1971, J. Lab. Clin. Med. 78, 316. Urbanitz, D., 1974, Klin. Wochenschr. 52, 542. Van Furth, R., and T.C. Van Zwet, 1973, in: Handbook of experimental immunology, Vol. 2, ed., D.M. Weir (Blackwell Scientific Publications, Great Britain), p. 36.1. Waithe, W.I., and K. Hirschhorn, 1973, in: Handbook of experimental immunology, Vol. 2 ed., D.M. Weir (Blackwell Scientific Publications, Great Britain), p. 25.7. Ward, P.A., 1968, J. Exp. Med. 128, 1201.
QUANTITATIVE ASSAYS OF HUMAN MONOCYTE-MACROPHAGE FUNCTION
WILLIAM L. WESTON, ROBERTA D. DUSTIN and STEVEN K. HECHT
Division of Dermatology, Department of Medicine, University of Colorado School of Medicine, Denver, Colorado, U.S.A. (Received 17 December 1974, accepted 17 February 1975)
Monocyte-macrophages are required for the development of cell mediated immunity to a variety of microorganisms and tumors. Quantitative assays of human monocytemacrophage function would be most useful in the evaluation of cell mediated immune function in man. Five quantitative assays are described that provide a human monocytemacrophage function profile. These assays parallel the physiologic steps necessary for monocyte-macrophages to function as phagocytes: 1) chemotaxis, 2) opsonization, 3) phagocytosis, 4) phagocytosis-induced metabolic stimulation and 5) destruction of foreign material. Application of these quantitative assays will allow detection and dissection of disorders of monocyte-macrophage function in man.
INTRODUCTION
Quantitative assays of human monocyte-macrophage function should take into account the physiologic mechanisms necessary for these cells to perform as phagocytes. These monocyte-macrophage functions can be divided into a series of physiologic steps, each of which can be quantitated: 1) cell movement, 2) adherence of particles to this cell surface, 3) engulfment (phagocytosis), 4) stimulation of intracellular metabolism and 5) destruction of foreign material. Five quantitative assays of human monocyte-macrophage function have been performed to measure these steps: 1) chemotaxis, 2) opsonization of the yeast particles, 3) phagocytosis of (a) paraffin oil and (b) bacteria, 4) phagocytosis-induced Nitro Blue Tetrazolium (NBT) reduction and 5) bactericidal assay for Staphylococcus aureus. This allows compilation of a monocyte-macrophage function profile in evaluating human host defenses. Stimulation or activation of monocyte-macrophages is a common, perhaps invariable, requirement for the development of cell mediated immunity (CMI) against both tumors and microorganisms (Bjorklund et al., 1972; Supported in part by NIH Research Grant, #AM 16752 Reprint requests to be sent to William L. Weston, Division of Dermatology, University of Colorado Medical Center, 4200 East Ninth Avenue, Denver, Colorado 80220.
214
Lejeune and Evans, 1972; Lohmann-Matthes, 1972; Evans, 1973; Nelson, 1974). There is an increasing awareness of the importance of the monocytemacrophage in host defense against tumors and microorganisms (Evans, 1973; Nelson, 1974). Quantitative assays of monocyte-macrophage function are essential for the evaluation of host defenses in a great variety of clinical settings. MATERIALS AND METHODS
Separation of rnonocytes Ten to 15 ml of heparinized (sodium heparin, 10 units/ml) venous blood was withdrawn from 20 healthy young adult donors, ages 25--35. The blood was divided into aliquots of 4 ml, each of which was layered over 1.5 ml of 9% Ficoll-Hypaque (sodium diatrizole) solution in 6-ml plastic test tubes after the technique of B o y u m (1968). The Ficoll-Hypaque solution contains 10 parts of Hypaque to 24 parts of Ficoll. After centrifugation at 400 g for 40 min a distinct layer of mononuclear leukocytes appeared which was removed carefully with a Pasteur pipette and transferred to a 12-ml plastic test tube. The cell suspension was washed three times with RPMI-1640 tissue culture media (Grand Island Biological Company, Grand Island, New York) and centrifuged at 200 g for 10 min. The cells were resuspended in 1 ml of RPMI-1640. Further purification of the monocytes was obtained by layering the cell pellet over a 28% bovine serum albumin (Path-O-Cyte 5, Miles Laboratories, Elkhart, Indiana) gradient and centrifuging at 400 g for 50 rain. The m o n o c y t e layer on t o p was aspirated and cell numbers were counted with a Unopette dilution system and a standard hemocytometer. Trypan Blue viability (Waithe and Hirschhorn, 1973) and neutral red phagocytosis (Gesner and Howard, 1967) were performed on the cells to determine the percentage of phagocytotic cells and their viability. All assays were adjusted for the percentage of phagocytic mononuclear cells in the final preparation.
Chemotaxis assay Chemotaxis was modified from the method of Boyden (1962). Stainless steel chambers of the Sykes--Moore variety were utilized and separated into two compartments by two Millipore filters. We used an 8.0-micron upper filter and 0.45-micron lower filter allowing measurement of any cells that may pass through the upper filter due to adherence defects and be entrapped on the lower Millipore filter (Keller et al., 1972; Frei et al., 1974). The standard chemotactic attractant used in our assay is a 10% solution of human serum in RPMI-1640 tissue culture media activated with E. coli lipopolysaccharide endotoxin type O l 1 1 : B 4 (Difco Laboratories, Detroit, Michigan) at 200 pg/ml. (Ward, 1968; Baum, 1971). This amount of endotoxin has been found to be the most effective in activating the C3 and C5
215 components of the complement system (Gewurz, 1968). All serum used in the assay was obtained from a single donor and used throughout the study giving a standard attractant. 0.5-ml aliquots of the serum were stored and frozen at --70°C. Attractants were added by injection into the lower chamber of the Sykes--Moore chamber. 1 X 106 m o n o c y t e s / m l were added to the upper c o m p a r t m e n t and all assays were performed in triplicate. Chambers were incubated at 37°C with 5% CO2 and 100% h u m i d i t y for 5 hr. After incubation the filters were removed and fixed in absolute methanol and rinsed in distilled water. Filters were stained using hematoxylin and blueing agents and then dehydrated in a standard fashion and cleared with xylene. Filters were dried overnight and then trimmed and m o u n t e d on glass slides with cover slips. Ten high power fields were counted. The cells on the upper surface of the 8.0-micron filter (cells that did not migrate) were compared to those migrating through the filter (including those within the filter) plus those that passed through the 8.0-micron filter and were entrapped on the surface of the 0.45-micron filter. The results were expressed as the percentage of total cells migrating by the formula: Total number of cells migrating Total number of cells n o t migrating
X 100
By expressing the results as the percentage of mononuclear leukocytes migrating, a means of comparison between investigators using the same procedures is provided and a precise number of cells does not have to be delivered to the upper membrane surface.
Monocy te phagocy tosis and phagocy tosis-induced NBT reduction The m e t h o d of Stossel (Stossel et al., 1972; Stossel, 1973) for simultaneous measurement of phagocytosis and NBT reduction by phagocytic cells was adapted for monocytes. This m e t h o d of phagocytosis was chosen because it avoids the adherence of phagocytic material to cell surfaces giving a false appearance of internalization of particles. Paraffin oil (Fisher Chemical Company, Fairlawn, New Jersey) does not adhere to cell surfaces (Stossel, 1973). The m o n o c y t e concentration was adjusted to 1 X 106 monocytes/ml. 500 mg of Oil Red O (Paragon C and C Company, Bronx, New York) was placed in a flask containing 0.5 ml of chloroform. This mixture was diluted to 20 ml with paraffin oil at a density of 0.89. These substances were mixed overnight and centrifuged at 2 0 0 g for 1 hr. The supernatant fluid was pooled in an Erlenmeyer flask and chloroform removed by evaporation overnight at 50°C under vacuum. It was again centrifuged at 2000 g for 1 hr. The optical density of the Oil Red O solution at 525 nanometers was used to compute a constant (K) for converting optical density of mg of paraffin oil ingested where:
216 1/O.D. K - - × 0.89 ml of dye Optical density of the final solution was found to undergo no change over a period of weeks (Stossel, 1973). The emulsion was prepared with 30 mg of E. coli lipopolysaccharide O l l l : B4 prepared by the Bouin procedure (Difco Laboratories, Detroit, Michigan). This was suspended in 3 ml of Hank's balanced salt solution (Difco Laboratories, Detroit, Michigan} in a 6-ml plastic test tube. The lipopolysaccharide was dispersed by vortex mixing for 15 seconds, then 1 ml of paraffin off containing Oil Red O was layered over the lipopolysaccharide suspension. The oil was emulsified into droplets by vortex mixing for 60 sec. Aliquots of this emulsion were frozen at --20°C. NBT was suspended in Hank's balanced salt solution at a concentration of 2 mg/ml and filtered through a 0.9.2 Millipore filter. Standardization was accomplished by adding 0.1 ml of the filtrate to 0.04 ml of 1 mM ascorbic acid and 0.2 normal sodium hydroxide. This reduced the NBT to formazan and the entire filter was dissolved i n 2 ml of p-dioxane with heating at 85°C and the optical density was determined immediately at 580 nm on a spectrophotometer. The conversion factor from the optical density of pg of formazan has been established by previous investigators as 14.14 tLg of formazan per optical density unit for 1 cm light path in dioxane at 25°C (Stossel, 1973). Serum, either used fresh or frozen at --70°C, was divided into 0.4 ml aliquots and incubated with 0.4 ml of the paraffin oil particles at 37°C for 15 min to opsonize the emulsion. A simultaneous assay was performed by adding 0.015 ml of 0.2 M potassium cyanide in Hank's balanced salt solution in two separate Falcon plastic test tubes that contained 0.4 ml of the leukocyte suspension. 0.4 ml of Hank's balanced salt solution was added to the first tube and 0.4 ml of the NBT solution was added to the second. After shaking in a water bath of 37°C for 5 min, 0.2 ml of emulsified Oil Red O particles opsonized by patients serum was added and the mixture incubated for 5 min at 37°C in the water bath. Six ml of ice cold 1 mM N-ethylmaleimide in 0.15 M sodium chloride was added and the uningested emulsion removed by centrifugation at 500 g for 15 min. The supernatant was discarded and the leukocyte pellet resuspended by adding 6 ml of fresh 0.15 M sodium chloride. The mixture was centrifuged at 5 0 0 g and the supematant discarded. Oil Red O and formazan were extracted from the pellets by adding 3 ml of p-dioxane and heating the extracts at 85°C for 15 min. Optical density solutions were clarified by centrifugation at 1000 g for 15 min and then determined at 525 nm and 580 nm. Results were expressed as mg of paraffin oil ingested per min per 107 monocytes. Results were calculated for the following formula: O.D.s2s X 50 Leukocytes/ml × m o n o c y t e s / 1 0 0 leukocytes
XK
217 NBT was expressed as pgm of formazan generated per min per 107 monocytes as derived from the following formula: O.D.ss0 in the extract with formazan -- O.D.ss0 in the extract w i t h o u t formazan X 50 X 14.14 Leukocytes/ml X monocytes/100 leukocytes
Monocyte attachment o f opsonized yeast Opsonization of yeast by the method of Miller (1969) was modified for monocytes. Preservative free bakers yeast was prepared by dissolving 100 ml of yeast in 8 ml of 0.9% NaC1 and incubating in a boiling water bath for 30 min to kill the yeast. The yeast was then filtered and the concentration was adjusted to 20 X 106 yeast particles/ml in a solution of 0.9% sodium chloride. 0.5 X 10 ~ monocytes/ml were mixed with 0.5 ml of AB negative h u m a n serum as an opsonin in a 6 ml plastic test tube. 0.05 ml of the 20 X 10 ~ yeast particle suspension was added to the mixture yielding a yeast : m o n o c y t e ratio of 1 : 5. The mixture was incubated at 37°C with 5% CO2 and 100% h u m i d i t y and aliquots removed after 5, 15 and 60 min of incubation with constant tilting and mixing. Thin layer smears were prepared of the aliquots and Wright's stained slides were counted and the number of yeast particles ingested/cell was determined as well as the percent of cells ingesting or attaching yeast.
Monocyte bactericidal assay and bacterial phagocytosis By utilizing the m e t h o d modified from Tan et al., (1971) it is possible to separate killing from phagocytosis. This is accomplished by the use of lysostaphin, a p o t e n t anti-staphylococcal agent that does not enter the cell. By destroying all the extra-cellular staphylococci one can measure only those organisms that have been engulfed. Defects in phagocytosis would otherwise masquerade as defects in killing because viable bacteria would remain on the cell surface. An inoculum of Staphylococcus aureus 502A was cultured overnight in trypticase soy broth. The bacteria was centrifuged and the pellet resuspended in 0.9% sodium chloride and centrifuged again at 50g. The bacterial pellet was diluted with Hank's balanced salt solution until the optical density at 550 nm was 0.3. This optical density corresponded to approx. 10 X 106 viable bacteria/ml. 10 X 106 monocytes in 1 ml were transferred with a siliconized Pasteur pipette and centrifuged at 200 g for 5 min. The supernatant was discarded and the pellet resuspended in 0.05 ml of sodium heparin and 0.5 ml of 0.1% Hank's gel (0.1% gelatin USP, J.T. Baker Company, in Hank's balanced salt solution, sterilized by autoclaving), with a siliconized sterile Pasteur pipette. One ml of AB negative human pooled serum was diluted with 4 ml of Hank's balanced salt solution to make a 20% opsonization solution. The solution was mixed for 5 min and 0.5 ml
218 o f this solution was added to 0.5 ml of the m o n o c y t e suspension. In a control tube m o n o c y t e s were o m i t t e d and 0.5 ml of Hank's balanced salt solution was added. 0.1 ml of t he bacterial suspension was added to each tube. The tubes were mixed gently and a 0.1 ml aliquot was removed and diluted in 10 ml of sterile distilled water. These were mixed for 30 sec and a fu rth er 1 to 100 dilution was made by diluting 0.1 ml of 10 ml distilled water. These were mixed by inverting and a 0.1 ml aliquot was pipetted and streaked over a trypticase soy agar Petri dish for a baseline (O) col ony count. All plates were assayed in duplicate. The tubes were then tightly capped and placed on a tilting table at 37°C incubation with 100% humidity for 2 hr. 0.2 ml o f each sample was transferred to a 6-ml plastic tube and 1 ml of filtered lysostaphin. 500 units/ml were added to each tube and placed in a 37°C water bath for 20 min. 0.1 ml was pipetted from each of the original mixtures o f cells and staphylococcal organisms and diluted in 10 ml of distilled water. These tubes were mixed for 30 seconds and a 0.1 ml aliquot was removed and rediluted in 10 ml of distilled water. In the patient test samples, 0.1 ml of this dilution was diluted in 2 ml of distilled water. The tubes were again mixed by inverting and 0.1 ml o f each tube was pipetted into trypticase soy agar plates, streaked and incubated at 37°C overnight. Similar dilutions were made for the lysostaphin after neutralization with 0.1 ml o f 2.5% trypsin and then incubated overnight in the bacterial incubator at 37°C. All plates were c o u n t e d using a view box after the overnight incubation. A percent of total viable bacteria (%TVB) was calculated by the following formula: %TVB =
No. colonies at 2 hr (2.1 X 104) No. colonies at 0 hr (1 X l 0 s)
The p er cen t o f intracellular bacteria (%IB) was calculated from the following formula: No. colonies at 2 hr after lysostaphin (1.05 X 104) %IB
=
No. colonies at 0 hr (1 X 105)
The p er cen t o f extracellular bacteria retaining (%EB) was calculated by the following formula: %EB
= %TVB
-- %IB
RESULTS The results are designed as a m o n o c y t e - m a c r o p h a g e funct i on profile and are expressed as the mean -+2 standard deviations as shown in the table. Chemotaxis is best expressed as the percent of total cells migrating as a standard frame of reference for o t h e r m e t ho ds of chemotaxis as suggested by
219 TABLE 1 Monocyte-macrophage function profile. Assay
Mean ± 2 S.D.
Chemotaxis (% cells responding)
11.62 +- 6.04
Opsonization (% cells with yeast adhering)
5 min 20.4 + 11.1 15 min 38 ± 21.6 60 min 43.7 + 38
Phagocytosis (pg paraffin oil ingested/min/107 monocytes) (% extracellular bacteria surviving)
0.153 ± 0.072 17.7
Intracellular metabolism (pg formazan reduced/minute/107 monocytes) 391 Killing (30 controls) (% intracellular bacteria surviving)
± 10.6 -+ 203
0.77 -+ 0.40
Naidu and Neubould (1974). A t t a c h m e n t appeared to show optimal differences after 15 min of exposure of opsonized yeast particles. At 60 min the variability was t o o great to permit distinction bet w een normal and abnormal states. The percent of total viable bacteria and the bactericidal assay is a useful screening procedure for bot h phagocytosis and the bactericidal assay. By calculating the pe r cent of intracellular bacteria surviving an accurate measure of those organisms phagocytized and killed is obtained. Defects in phagocytosis would otherwise mask these results and result in a faulty d e t e r m i n a t i o n o f defective intracellular killing o f bacteria (Tan et al., 1971; Van F u r th and Van Zwet, 1973). DISCUSSION Despite the i m p o r t a n c e of m onocyt e- m acrophages in host defenses against infections and t um or s there has been a paucity of reports on m o n o c y t e macrophage f u n c t i o n in humans. Some investigation of m o n o c y t e function in humans has been directed toward in vitro chemotaxis (Ward, 1968; Snyderm an et al., 1972) and in vivo cell m o v e m e n t (Meuret and H of f m a n, 1973). Defective m o n o c y t e chemotaxis has been det ect ed in patients with cancer (Snyderm an et al., 1974; S n y d e r m a n and Stahl, 1974) and a child with chronic moniliasis (Snyderman, 1973). M o n o c y t e phagocytic f u n c t i o n f or IgG-coated e r y t h r o c y t e s (Huber, 1968) has been investigated and shown to be diminished by Rifampin (Urbanitz, 1974). Phagocytosis of bacteria has been f o u n d to be normal in children
220 with neutropenia and chronic infections (Baehner and Johnson, 1972) and in m y e l o m o n o c y t i c leukemia (Cline, 1973) and lymphoma. Monocyte bactericidal activity was normal in children with neutropenia (Baehner and Johnson, 1972) but defective in leukemia and l y m p h o m a (Cline, 1973). A candicidal assay for m o n o c y t e s has been developed as well (Lehrer and Cline, 1969). Simultaneous m o n o c y t e functions have rarely been reported in humans (Baehner and Johnson, 1972; Cline, 1973) and then only for phagocytosis and killing b u t n o t for cell movement. More attention has recently been focused on the monocyte-macrophage and its role in host defense. Monocytes synthesize a large number of biologically important products including transferrin, complement, interferon, pyrogens and granulopoietin (Lobuglio, 1973). It has recently been postulated that impaired activity of the m o n o c y t e may explain a number of unusual infectious diseases seen in the newborn (Gotoff, 1974). The importance of the macrophage in clinical disease states (Cline, 1973; Meuret and Hoffmann, 1973; Snyderman, 1973; Snyderman et al., 1974; Urbanitz, 1974) and in t u m o r immunology (Evans, 1973; Nelson, 1974) from early studies suggests we are at the threshold of understanding the role of the macrophage in host defenses. The quantitative monocyte-macrophage assays described here offer several advantages in investigation of the m o n o c y t e ' s role in host defense. First, relatively small amounts of blood are needed to obtain this profile provided the subjects studied have normal peripheral white counts and often 7--10 ml of blood will suffice in children with elevated white counts whereas leukopenic patients may require large volumes of blood (30--40 ml). Further microquantitation of these assays will be required to facilitate investigation of leukopenic subjects. Second, preliminary cost analysis revealed that this complete profile costs $30.00 per subject excluding technician salaries. Laboratory equipment needed includes a centrifuge, bacterial incubator and tissue culture incubator in addition to the usual laboratory equipment (microscope, balance, etc.). An available autoclave also reduces costs. Thirdly, a maximum of 4 profiles can be performed in a day. It takes approx. 2 hr to separate blood and an average of 4 hr for most assays. Assays can be completed within an 8-hr working day. It would appear that 3 different assays of phagocytosis are included in the monocyte-macrophage function profile. However, only one of these assays measures phagocytosis; the others are included to measure other functions that are closely related to phagocytosis. Phagocytosis of bacteria must be included in the bactericidal assay so that one can distinguish between faulty killing and faulty phagocytosis within that particular assay. If bacteria were not ingested they could not be destroyed, resulting in a high percentage of viable bacteria remaining leading to the false conclusion that it was measuring faulty killing. The most accurate measurement of phagocytosis is that of
221
paraffin oil ingestion since paraffin oil does not adhere to cell surfaces and all measured is that ingested. The third assay, utilizing opsonized yeast, really measures attachment of yeast since there is no method to distinguish between the yeast on the m o n o c y t e surface and that ingested. It is incorrect to consider this assay a phagocytic assay and it is included as an assay to measure attachment to the m o n o c y t e surface. Substituting another assay such as surface receptors for IgG or a cytotoxicity assay may be desired by other investigators. Development of these quantitative assays of human monocyte-macrophage function which take into account the physiologic mechanisms of these cells acting as phagocytes will provide the data to define the role of the monocyte-macrophage in host defenses.
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222 Tan, J.S., C. Watanakunakorn, J.P. Phair, 1971, J. Lab. Clin. Med. 78, 316. Urbanitz, D., 1974, Klin. Wochenschr. 52, 542. Van Furth, R., and T.C. Van Zwet, 1973, in: Handbook of experimental immunology, Vol. 2, ed., D.M. Weir (Blackwell Scientific Publications, Great Britain), p. 36.1. Waithe, W.I., and K. Hirschhorn, 1973, in: Handbook of experimental immunology, Vol. 2 ed., D.M. Weir (Blackwell Scientific Publications, Great Britain), p. 25.7. Ward, P.A., 1968, J. Exp. Med. 128, 1201.
