Quantitation of the rate of ingestion of Escherichia coli by human polymorphonuclear neutrophils using [3H]uracil

223

Journal o[ Mu'robu~logtcal Method~ 3 (1985) 223 235

Elsev,er J M M 00099

Quantitation of the rate of ingestion of Escherichia coli by human polymorphonuclear neutrophils using [3H]uracil Paul S. Cohen*, Renate SoIL Josef Suter and Klaus Vosbeck** Research Department, CIBA-GEIG Y Limited. CH-4002 Basel (Swttzerland)

(Recewed 12 October 1984) (Rewsed version received 3 January 1985) (Accepted 7 January 1985)

Summary A method using [3H]uracd to simply and accurately measure the rate of ingestion of E. coli by human polymorphonuclear neutrophils (PMN) is described. The method is based on the observation that [~H]uracil penetrates bacterial membranes and is incorporated into RNA, whereas it is not taken up to a s~gmficant extent by PMN. Therefore, extracellular bacteria can be selectively labeled during pulses with [3H]uracd whereas phagocytized bacteria are not labeled. Using this pH]uracil incorporation method along with viable count determinations, it was shown that although about 5% of ingested E. coli B-1385 remained viable for at least 3 h after being ingested by PMN, the surviving bacteria may have become damaged as shown by a decreased abihty to incorporate [3HIuracil into RNA when released from PMN. Simdarly, it was shown that PMN treated with cytochalasin B continued to ingest E. coil B-1385, but the bacteria were not kdled and, m fact, continued to grow. This method, together with other techniques of assessing bacterial phagocytos~s, may therefore be useful for a more detaded analysis of the process of bacterial phagocytosis than was hitherto possible.

Key words

Bacteraal viabthty - Cvtochalasin B - Phagocvtosis - Polymorphonuclear neutrophils labelling -Uractl mcorporatton

Radio-

Introduction Polymorphonuclear neutrophils (PMN) bind, ingest and kill opsonized bacteria. In several studies attempts were made to separately measure the ingestion phase and intracellular killing of bacteria [1, 2]. In these studies either antibiotics or lysostaphin was used to kill extracellular Staphylococcus aureus, or PMN and extracellular bacteria were separated by centrifugation [3-7]. There is still controversy as to whether the drugs * On leave from the Umverstty of Rhode Island, Department of Microbiology, Kingston, RI 02892, USA. ** To whom all correspondence should be addressed. 0167 7012:85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Dwtslon)

224 used to eliminate extracellular bacteria bind to and penetrate PMN [1], and separation of PMN and extracellular bacteria by centrifugation does not permit a rapid determination of extracellular bacteria. Yamamura et al. [8] made a significant contribution toward this end by devising an elegant, yet simple method to measure phagocytosis by PMN as inhibition of radioactive uridine uptake by Candida albwans. This method was based on the fact that yeast cells, but not P M N incorporate uridine. Therefore ingested yeast cells should not incorporate radioactive uridine. Subsequently, Lam and Mathison [9] adapted the method to the study of phagocytosis of Staphylococcus aureus. While in each of these studies it was proven conclusively that PMN incorporated little, if an3', labeled uridine, and that the ability of both Candtda albicans and Staphylococcus aureus to incorporate rad~oactive uridine decreased when exposed to PMN, it was not proven directly that the loss in uridine incorporating ability was due solely to ingestion of the microorganisms by PMN. Furthermore, the labeling of either Candida albicans or Staphylococcus aureu,~ with radioactive uridine did not exceed 104 cpm in one hour at 37°C, making it difficult to study the kinetics of phagocytosis. By using [3H]uracil of high specific activity to label E. coli in a medium which supported bacterial growth, we were able to study the kinetics of ingestion of bacteria by human PMN, since extracellular bacteria incorporated more than l X l05 cpm into RNA within 5 min. Under these conditions, we were able to show directly that only E. co# which were not ingested by human PMN in phagocytosis mixtures were able to incorporate [3H]uracil. Furthermore, by combining this [3H]uracil assay with viable count measurements we have found that not all ingested E. coli are killed and that those which survived may become damaged. Materials and Methods

Med~a Dulbecco's phosphate buffered saline containing CaC12 (0.1 g/l) and MgCI2-6H,O (0.1 g/l) was supplemented with 1 g/'l of D-glucose (PBS-G). PBS-G containing 10% fresh human serum obtained from a single donor and kept frozen at -70°C (PBS-G-S) was used for opsonizing bacteria. For all phagocytosis and other labeling experiments, PBS-G containing 1% human serum, 0.1 g/l MgSO4.7H20, 1.0g/l (NH4)_,SO4 and 0.6 g/1 casamino acids (Difco) was used (PBS-G-A). Bacteria Escherichia coli B-1388 (09: K30+), E. coli B-1385 (09: K30, E. coli B-1374 (018:K5 +) and E. coliB-1375 (018:K5) were obtained from K. Jann, Max-Planck-lnstitut ftir lmmunbiologie, Freiburg, W. Germany. The strains were kept frozen in liquid nitrogen. For use, they were maintained on BHI agar plates. Bacteria were grown overnight m trypticase soy broth (TSB; Oxoid), washed once in PBS-G and subsequently adjusted photometrically to the appropriate density in the required medium. Bacteria were preopsonized, if required, for 10 min at 37°C at a concentration of l08 bacteria/ml in PBS-G-S. Thereafter they were diluted into PBS-G-A to a concentration of 5× 107 bacteria/ml and preincubated on a rotator (4 rpm, 37°C) for 25 rain.

225 Polymorphonuclear neutrophils ( P M N) Forty ml of citrated blood from one healthy human donor were mixed with 20 ml of physiological saline containing 60 g/1 of Dextran T70 (AB Pharmacia, Uppsala, Sweden) and the erythrocytes were allowed to sediment for 60 rain at 37°C. The leukocyterich supernatant was centrifuged (200Xg, 10 min. 20°C) and the sediment resuspended for 10 min in 7.8 g/1 NH4C1 to lyse remaining erythrocytes. The leukocytes were washed twice in PBS-G (200Xg, 10 min) at 20°C and adjusted to the appropriate cell density in the required medium. Cell numbers were determined using a hemocytometer. This procedure yielded a preparation consisting of 80-85% PMN, 10-15% lymphocytes and 5% monocytes. Phagocytosis assay - determination o f viable counts A suspension containing 107 PMN per ml and 5 x 106 preopsonized and preincubated bacteria per ml in PBS-G-A was prepared. Aliquots of 1.0 ml were distributed into small polypropylene tubes (Eppendorf, Hamburg, W. Germany) and rotated end-overend at 37°C (4 rpm). After the indicated incubation times aliquots were either diluted directly (intact PMN) or the sample was centrifuged (10000Xg, 5 min) and the pellet resuspended in 1.0 ml distilled water for 15 min at 20°C and then diluted (lysed PMN) in calcium- and magnesium-free PBS containing 2 mg/1 of phenol red using microtitration equipment (Titertek, Flow Laboratories, Baar, Switzerland). Aiiquots (20 #l) of the dilutions were placed on Brain Heart Infusion (BHI; Oxoid) agar and the number of viable bacteria was calculated from the number of colonies counted after overnight incubation at 37°C. Phagocvtosis assay - pulse labe#ng with [3H]uracil Aliquots (1.0 ml) of the same mixture of bacteria and PMN that was used to measure viable bacteria were distributed into 15 ml polystyrene tubes (Falcon; Becton-Dickinson, Basel, Switzerland) and rotated end-over-end (4 rpm) at 37°C. At the specified incubation times, 10#1 of a solution of [6-3H]uracil (100#Ci/ml, 25 mCi/mmol) in 70% ethanol (New England Nuclear, Boston, MA, USA) was added. After 5 min of incubation at 37°C the incorporation was stopped by adding 1.0 ml of ice-cold 10% trichloroacetic acid (TCA), and the samples were put on ice. Thereafter 5 ml of 5% TCA containing 1 mg/ml of uracil were added and the samples were filtered through Whatman G F / C glass fibre filters (Whatman Ltd., England). The filters were washed four times with 5 ml of cold (4°C) 5% TCA containing I mg/ml of uracil, dried and counted by liquid scintillation counting using toluene containing 5 g/l of 2,5-diphenyloxazole (PPO) and 0.3 g/l of 1,4-bis-2-(5-phenyloxazolyl)-benzene (POPOP) as scintillation fluid. Radioactivity counts were quenched by 50% in the presence of PMN. Results were corrected for this quenching effect where appropriate. Transmission electron microscopy To the suspension of PMN and bacteria (l.0 ml) 0.5 ml of cacodylate buffer (0.1 M sodium cacodylate, pH 7.4) containing 3% glutaraldehyde were added at the appropriate time and the tubes were left in crushed ice for 30-60 min. Thereafter, the samples were centrifuged (750Xg, 10 min) and the supernatant was replaced by fresh cacodylate

226 buffer containing 3% glutaraldehyde. After 20 h. the sediment was fixed for 2 h with I% osmium tetroxide in 0.18 M sodium cacodylate buffer, pH 7.4, and after dehydration in graded acetone, embedded in Epon resin [10]. After polymerization, the blocks were sectioned with a Reichert-Jung 'Ultracut' ultramicrotome, the sections bemg placed on 200-mesh copper grids fitted with a carbon-coated collodion film. Contrast was enhanced by double-staining with uranyl acetate and lead citrate [Ill. The sections were examined in a Philips EM-300 instrument, and micrographs were recorded on 6.5X9 cm films.

Chemicals Cytochalasin B (Sigma Chemical Co., St. Louis, MO, USA) was dissolved in D M S O and diluted to a final concentration of less than 0.1% D M S O in the required media. All chemicals used were of reagent grade. Results

Labeling of E. coli B-1385 with [3H]uracil E. coli B-1385 incorporated [3H]uracil (I #Ci/ml, 4 . 6 × 1 0 3 #g/ml) into R N A in PBS-G-A in proportion to the concentration of preopsonized bacterial cells between 5X 103 and 5X 106 bacteria per ml (Fig. 1) and labeling was linear for at least 15 rain at 5X106 bacteria per ml (Fig. 2). Furthermore, E. coliB-1385 incubated in PBS-G-A incorporated the same amount of [3H]uracil into R N A per viable cell as long as the bacteria remained in the exponential phase of growth (data not shown). Routinely, P M N and opsonized E. coliB-1385 were mixed at 1)<107 per ml and 5X10 ~ per ml, respectively, and incubations were for no longer than 3 h. Pulses with [3H]uracil were for 5 rain at various times after preopsonized E. coli B-1385 and P M N were mixed.

Measurement of phagocvtosis by pulse labefing with [3H]uracil Preopsonized E. coliB-1385 rapidly lost the ability to incorporate [3H]uracil into R N A when mixed with P M N (Fig. 3). In contrast, when E, coil B-1385 was incubated log cpm 6i

.y./"

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3 2

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1

log cfu Fig I. lncorporatton of [~H]uracd (tog cpm) as a functton of bacterial concentration (log cfu) The indicated concentrations of preopsomzed E coh B-1385 were pulse-labeled with [-~H]uracil for 5 mm as described m Materials and Methods.

227

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200000

I000001 ~~" /° k', 10 1'5 Pdse trine (ram)

Fig. 2. incorporation of [3H]uracd into RNA of E. coli B-1385. Preopsonized E. coliB-1385 (5x 10~/ml) were preopsonized and incubated in the presence of [~H]uracil (1 pCi/ml, 4.6x 10 3 ~ug/ml) for the indicated times (pulse time). Thereafter samples were processed and the radioactivity incorporated into RNA (cpm) was determined as described in Materials and Methods.

in the absence of PMN, its ability to incorporate [3H]uracil increased slowly as it grew (Fig. 3). P M N alone incorporated [3H]uracil at a rate of less than one percent of that of the bacteria (Fig. 3). Identical rates of loss of the ability of E. coli B-1385 to incorporate [3H]uracil were shown to occur with either one or 5 min pulses, but the 5 min pulse allowed greater sensitivity of the assay. cpm 105

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30 6'0 9'0 Incubatmnt=rne(ram) Fig. 3. [~H]uracil incorporauon as a measure of phagocytosis. Preopsonlzed E. coli B-1385 (5× 106/ml) and PMN (107/ml) were mixed and incubated at 37°C. At the indicated times, samples were pulsed for 5 min with [3H]uracil (1 pCi/ml, 4.6× 10 3 pg/ml). Thereafter samples were processed and the radioactivity incorporated into R N A was determined as described in Materials and Methods. The cpm were corrected for the 50% quenching caused by P M N constituents. (e) E. coli B-1385 and PMN; (0) E. cob B-1385 alone; (z~) PMN alone.

228

Relationship between the rate off [3H]uracil incorporation and the fraction of free E. coil B-1385 The question remained as to whether bacteria not associated with P M N were the only source of [3H]uracil incorporation, or whether a significant proportion of [3H]uracil incorporation occurred in bacteria bound to the outer surface of PMN. P M N and E. coli B-1385 were therefore mixed and at various times of incubation at 37°C samples were centrifuged (I lO×g, 4 rain) to remove P M N . Under these conditions, 96-98% of the P M N were removed while a bacterial suspension centrifuged identically showed no detectable loss of viable count (data not shown). The bacteria in the supernatant, as well as in a control sample that had not been centrifuged, were pulsed with [3H]uracil and assayed by viable count (Fig. 4). Clearly, the loss of [3H]uracil incorporation in centrifuged and uncentrifuged samples were nearly identical, suggesting that [3H]uracil incorporation took place almost exclusively in free bacteria and not in bacteria which were in any way associated with P M N . Furthermore, in support of this view, the loss of [3H]uracil incorporation was directly proportional to the loss of viable count in the centrifuged sample at all times (Fig. 4). The degree to which E. coli B-1385 was ingested by P M N was also examined after various times of incubation with P M N by phase contrast microscopy of semi-thin sections and by transmission electron microscopy (Fig. 5). In order to avoid artefactual alterations of the spatial relationship between bacteria and phagocytes by centrifugation and processing of the samples, the assay mixtures were first fixed and then processed at 0°C. Vertical semi-thin sections of the sediments were inspected and quantitatively evaluated at several levels to rule out the possibility of artefacts due to the formation of gradients of bacterial and cellular density. At all times only a negligible proportion (< 1%) of bacteria was found to be bound to the external surface of P M N . It therefore

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Fig 4. [~H]uracd mcorporauon into free and PMN-assoctated bacteria. E. coh B-1385 was preopsomzed and incubated with PMN (10T/ml) at 37°C. At the indicated times PMN were selectivelyremoved by centnfugatlon (1 lO×g, 5 mm). The number of viable bacteria (O) and the [3H]uracil incorporation (e) were measured m the suspensmn before centrffugatmn (--) and in the supernatant after centrifugatlon (--). The initial values for the number of viable bacteria and for the [~H]uracil incorporation were 3.5× 10~/ml and 57 264cpm, respectively

229

4D

/

Fig. 5. Intracellular localization of E. coli B-1385, The fraction of extraceUular preopsonized bacteria after incubation with PMN for 90 rain was estimated by transmission electron microscopy of thin sections. PMN and bacteria were incubated at a ratio of I : 20 for these experiments. The bar represents 2/~m.

230 appears that ingestion occurred almost immediately after collision between E. coh B-1385 and the PMN, and that loss in the ability of bacteria to incorporate [3H]uracil was due exclusively to being ingested by PMN.

Opsonization requirement for loss of[3 H]uracil incorporating abifity In general, bacteria must be opsonized with serum in order to be ingested and killed by P M N [12, 13]. To determine whether E. coli B-1385 must be opsonized to lose the ability to incorporate [3H]uracil into RNA when added to PMN, one half of a culture of E. coli B-1385 was preopsonized in PBS-G-S, and the other half was incubated identically in PBS-G. Each portion was then washed in PBS-G and resuspended at 5× 107 cells/ml in PBS-G-A without serum. The bacteria (5 × 106/ml, final concentration) were mixed with PMN prepared in the absence of serum (1 ×107/ml, final concentration). Opsonized E coil B-1385 were phagocytized as measured by both [3H]uracil incorporation and viable counts. In contrast, unopsonized E. coli B-1385 were not phagocytized as measured by either method (Fig. 6). Phagocytosis of encapsulated and unencapsulated E. coli strains Certain encapsulated E. coli strains, in contrast to unencapsulated mutants derived from them, are not readily phagocytized in one percent serum [14]. Two pairs of isogenic encapsulated and unencapsulated E. coli strains (E. coli 09: K30+/E. coli 09: K30 [= E. coli B- 1385] and E. eoli 018 : K5 + / E. coli 018 : K5-) were preopsonized, and their interaction with phagocytes was followed by [3H]uracil incorporation and viable counts (Fig. 7). As measured by either method, the encapsulated strains were not phagocytized, whereas the unencapsulated strains were readily phagocytized.

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F,g. 6 Phagocytosis of opsomzed and unopsomzed E. coh B-1385. (A) [~H]uracil ,ncorporauon. (e) preopsonlzed E. coh B-1385 and PMN; (O) unopsomzed E. coliB-1385 and PMN. The values of 100% were 151099cpm for opsomzed and 159045 cpm for unopsomzed bacteria, respectwely (B) Viable counts. (e) preopsomzed E. coli B-1385 and PMN; (O) unopsonized E. coh B-1385 and PMN. The value of 100% was equivalent to 3.3X 106 preopsomzed E. coli B-1385 per ml and 2.5x 106 unopsonized E. coli B-1385 per ml.

231 Inhibition o f phago~3'tosis by cvtochalasin B

It has been reported that cytochalasin B inhibits monocyte mediated hemolysis of group A red blood cells, presumably by interfering with microfilament formation and funcuon, thereby inhibiting phagocytosis [15]. To further understand the action of cytochalasin B in the phagocytic process and to further explore the usefulness of the [3H]uracil phagocytosis assay, E. coli B-1385 was incubated for 90 min with P M N that had been pretreated with various concentrations of cytochalasin B ranging from zero to 10tag/ml for 30 min at 37°C. At that time, both [3H]uracil incorporation and viable counts were determined. As the cytochalasin B concentration increased, fewer E. coil B-1385 were killed, to the point that at 5 tag/ml cytochalasin B the viable count was identical to the initial viable count, and at 10 tag/ml a doubling in the original number of bacteria per ml was observed (Fig. 8). In contrast, even at the highest concentrations of cytochalasin B, [3H]uracil incorporation was no greater than 50% of that observed in the absence of PMN, indicating that 50% of the input bacteria were still ingested. Neither the D M S O in which the cytochalasin B was dissolved nor the cytochalasin B itself inhibited incorporation of [3H]uracil into E. coli B-1385 (data not shown). I m p a i r m e n t of[3H]uracil incorporation by E. coli, without death, caused by P M N During the course of this work, it became clear that not all ingested E. coli B-1385

were killed by PMN. This was shown directly during normal phagocytosis by comparing viable counts before and after selective removal of P M N from phagocytosis mixtures (Fig. 4). In fact, in the experiment illustrated in Fig. 4 at 150 rain after E. coli B-1385 and P M N were mixed, approximately 99% of the bacteria were ingested, but only 50% of the bacteria were killed. To determine the metabolic state of the ingested E.

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Fig. 7. Phagocytos~sof encapsulated and unencapsulated E. coli stratus E. coil stratus were preopsonized and mixed with human PMN (107/ml). (A) [~H]uracd incorporation. The value of 100% was equivalent to 128386 cpm for E. coli 018 : K5+, 132188cpm for E. coil 018: K5 , 148373cpm for E. cob 09: K30+, and 54913 cpm for E. coli 09:K30 (= E. cob B-1385). (B) Viable counts. The value of 100% was equivalent to 4.5x 106/ml for E. coli 018:K5+, 2.7x 106/ml for E. cob 018:K5 , 6.0X 106/ml for E. coh 09:K30+, and 2.8XI06/ml for E. coli 09:K30 (=E. coli B-1385). (e) E. coli 018:K5+; (0) E. coli 018:K5 ; (A) E. coil 09: K30+; (A) E. ('oh 09:K30 (= E. ('oh B-1385).

232 % of value at t=O mm //

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Fig. 8. Inhibit]on of phagocytoms by cytochalasm B, E. co//B-1385 was preopsomzed and mixed with PMN (1071ml) which had been premcubated with the indicated concentrations of cytochalasm B for 30 mm at 37°C The viable count and the [3H]uracil incorporation at the beginning of the experiment were 4,3x l0 b per ml and 153564 cpm. respectively. As an expression of the inhibition of phagocytosm, the relative values of the number of viable bacteria (O) and of the [3H]uracil incorporation (e) after incubation at 37°C for 90 mm are indicated.

coli B-1385 which remained alive, ingested bacteria were released by osmotically ruptur-

ing the P M N in water at 120, 150 and 180 min after phagocytosis had begun, additions were made to the water to make it equivalent to PBS-G-A, and the ability of the released bacteria to incorporate [3H]uracil into RNA was determined. Viable count measurements were also made. As illustrated in Fig. 9, the loss in [3H]uracil incorporaTing ability accurately reflected the loss in viable count during the first 60 rain of phagocytosis. However, after that time, whereas viable counts remained relatively constant at about 6% of the input bacterial concentration the [3H]uracil incorporating ability continued to decrease an additional ten-fold. When the bacteria were released at 120 mln, their ability to incorporate pH]uracil increased only three-fold rather than the ten-fold predicted if the ingested bacteria were unchanged. Furthermore, at 150 and 180 rain, impairment of [3H]uracil incorporation continued, since the viable released bacteria gained less than 10% of the ability to incorporate [3H]uracil into RNA which would have been observed had they been unchanged (Fig. 9). Control experiments showed that when E, coli B-1385 were subjected to the same conditions in which PMN were broken and were then incubated in the presence of P M N extract, they remained alive and continued to incorporate [3H]uracil into RNA at an undiminished rate (data not shown). It should also be mentioned that P M N preparations were found to be variable, i.e., in a second experiment bacteria released from PMN at 120 min incorporated

233 ~3.o

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incubatmnt)me {mm) Fig. 9. Damage of ingested E. coh B-1385. E. coil B-1385 was preopsomzed and incubated at 37°C with PMN (107/ml). At the indicated ttmes [~H]uracil incorporation (e) was measured, and the number of viable bacteria (©) was determined. In addition, at the indicated umes, the water was made equwalent to PBS-G-A and the released bacteria were pulse labeled with [3H]uracil. (zx. . . . A) ratio of [3H]uracd incorporated into released E. coli B-1385 to that incorporated into ingested E. coil B-1385. The initial values for the number of viable bacteria and [3H]uracil incorporation were 2.9 × 106/ml and 125 800 cpm, respectwely.

[3H]uracil into RNA as expected for free bacteria. However, a greater than three-fold decline in this ability was observed over the next hour (data not shown). Discussion The [3H]uracil phagocytosis assay reported here offers several advantages over the one devised by Yamamura et al. [8] for measuring phagocytosis of Candida albicans and used by Lam and Mathison [9] to measure phagocytosis of Staphylococcus aureus. These authors measured total radioactive uridine incorporated into the bacteria including the nucleoside itself and the uridine contained in RNA. That is, after a suitable incubation period in the presence of radioactive uridine, the bacteria and PMN were immediately collected on filters, washed and counted. In the present investigation, following 5 min pulses with high specific activity [3H]uracil, the reactions were stopped with TCA. The radioactive RNA was subsequently collected and washed as a precipitate on filters and then counted. This procedure has two advantages. First, TCA precipitation is a rapid procedure and the radioactive precipitates need not be filtered immediately, so that large numbers of samples can easily be handled. Second, the problem of bacteria leaching low molecular weight radioactive compounds during filtration is eliminated since only radioactive precipitates are retained on the filters. The measurement of true macromolecular synthesis as opposed to uptake of radioactivity in any form would, in addition, appear to yield more accurate data. It should also be noted that [3H]uracil rather than [~H]uridine or [14C]uridine was used in the present studies. The free base is far less expensive than the nucleoside and, in general, it is readily transported across bacterial membranes. The [3H]uracil phagocytosis assay specifically detected free E. coli B-1385. This was

234 shown directly by the fact that [3H]uracll pulses into RNA were identical in untreated phagocytosis mixtures and in mixtures m which PMN were selectively removed bx centrifugation (Fig. 4). Moreover, as revealed by transmission electron microscop), ingestion of E. coli B-1385 seemed to be ~mmediate since very few. if an,,', bacteria were seen to be adherent to the PMN surface once contact between the bactermm and PMN had been made (Fig. 5). It is well known that bacteria are phagocytized poorly by PMN in vnro if the)' are not opsonized as measured by conventional techniques [12, 13]. Opsonization was also required for ingestion of E. coli B-1385 by human PMN as measured by the [~H]uracil phagocytosis assay (Fig. 6). Therefore, ingestion of E. coli B-1385 by PMN as measured by the [3H]uracil assay agrees with results obtained by more conventional techniques, including viable counts (Fig. 6). Horwitz and Silverstein [14] demonstrated that in the presence of one percent normal human serum and PMN, encapsulated E. coli serotype 09:29~-: H was not ingested by PMN, but an isogenic nonencapsulated mutant was readily ingested. Phagocytosis was assayed in this study using phase-contrast microscopy. The [3H]uracil phagocytosis assay yielded the same results with two pairs of isogenic encapsulated and nonencapsulated E. coli strains (K5 and K30). The encapsulated strains were not ingested but their isogenic nonencapsulated mutants were readily ingested (Fig. 7). The [3H]uracil phagocytosis can be performed simultaneously with viable count measurements. Such analysis yields information that neither assay alone can provide. For example, it was found in the experiment that PMN both ingested and killed E. coil B-1385 at the same rate for 60 min (Fig. 9). However, after 60 min, PMN continued to ingest the bacteria at an undiminished rate, but failed to kill them (Fig. 9). Furthermore, it appeared that after 120 min, the ingested bacteria may have become damaged in an unknown way, as shown by their continually increasing loss of ability to incorporate [3H]uracil into RNA following release from PMN (Fig. 9). As a second example, cytochalasin B treatment of P M N inhibited ingestion of E. coli B-1385 as measured by the [3H]uracil phagocytosis assay. However, inhibition was incomplete, reaching a maximum of only 50% (Fig. 8). Furthermore, at a cytochalasin concentration of 10#g/ml, the highest concentration used, bacteria were not killed and, in fact, doubled in number (Fig. 8). Since only 50% of the original bacteria were not ingested by PMN, the bacterial growth observed took place inside the PMN. In summary, the [3H]uracil phagocytosis assay appears to be the one of choice when the rate of ingestion of bacteria by PMN is to be determined. It is more rapid and more accurate than measuring ingestion of prelabeled bacteria because the need to centrifuge and wash the PMN is eliminated. Moreover, the results of preliminary experiments presented here show that using the [3H]uracil assay along with simultaneous viable counts should lead to a better understanding of how PMN ingest, damage and kill bacteria.

Acknowledgements We thank our colleagues of the CIBA-GE1GY Health Service for their invaluable help in obtaining blood samples and Mrs. A. Kittl for excellent secretarial help.

235 References 1

2 3 4 5

6 7

8 9 10 lI 12

13 14 15

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