INCT ON AND IMMuNITY, Aug. 1978, p. 381-386 0019-9567/78/0021-0381$02.00/0 Copyright i 1978 American Society for Microbiology

Vol. 21, No.2

Printed in U.S.A.

Polymorphonuclear Neutrophil Chemotaxis Under Aerobic and Anaerobic Conditions DENNIS A. CASCIATO,",2 LEONARD S. GOLDBERG,2 AND RODNEY BLUESTONE 12* Medical and Research Services, Veterans Administration Hospital Center, Los Angeles, California 90073,' and Department of Medicine, University of Caifornia at Los Angeles Center for the Health Sciences, Los Angeles, California 900242 Received for publication 26 August 1977

The motility of human polymorphonuclear neutrophils was studied in vitro under aerobic and anaerobic conditions. Chemotactic factors were generated from plasma with immune complexes or with whole bacteria (Staphylococcus aureus, Escherichia coli, and Bacteroides fragilis). Chemotaxis induced by chemotactic factors generated from immune complexes was identical under both conditions. However, chemotaxis utilizing chemotactic factors generated from bacteria was markedly depressed under anaerobic conditions. Mean random tube motility was not significantly different under aerobic and anaerobic conditions. These data indicate that different metabolic pathways may be involved in polymorphonuclear neutrophil movement. Some of these pathways require oxygen (chemotaxis in response to factors generated by bacteria in plasma), whereas others do not (random tube migration and chemotaxis in response to factors generated by immune complexes in plasma). These observations may be important in the induction of inflammatory responses within hypoxic tissues.

Once polymorphonuclear neutrophils (PMNs)

enter the circulation, their physiological functions include movement into tissues, phagocytosis, and degradation of effete or foreign objects. At least two types of neutrophil movement are recognized, random migration and chemotaxis. Random migration is nondirected cell movement; chemotaxis is the directed migration of cells toward a stimulus. A great deal has been learned about the metabolic activities of PMNs during phagocytosis and bacterial killing (10). Much of this information has evolved from experiments which found that neutrophils could phagocytize, but could not kill, microorganism under anaerobic conditions (12). Similar information regarding oxygen requirements for neutrophil locomotion is lacking. We performed experiments to study the ability of PMNs to move and to respond to chemotactic stimuli in the presence and absence of oxygen.

MATERIALS AND METHODS Neutrophil suspensions. Blood was collected in heparinized tubes from 17 healthy human volunteers. After the erythrocytes were lysed with 0.8% ammonium chloride, the resultant leukocyte suspension was washed with Hanks balanced salt solution (HBSS) and adjusted to a final concentration of 5 x 106 PMNs/ml with HBSS containing 10% fetal calf serum. Bacterial suspenions. Three microorganisms were utilized, Staphylooccus aureus 502A and labo-

ratory reference strains of Escherichia coli and Bacteroides fragilis subsp. firagilis. All organisms were grown overnight at 370C, S. aureus and E. coli in tryptic soy broth in air and B. fragilis in Schaedler agar in GasPak jars (Baltimore Biological Laboratory, Cockeysville, Md.). The organisms were washed three times with HBSS and adjusted to an optical density at 650 nm of 1.00 with a Gilford spectrophotometer. CF suspensions. Chemotactic factors (CF) were generated from plasma of the respective PMN donors, using each of the three bacteria and immune complexes. Immune complexes were prepared from commercial human albumin (5 g/dl) and rabbit anti-human albumin (Miles Laboratories, Inc., Kankakee, Ill.) in proportions of 1:2. Suspensions of these CF stimulants were incubated with equal volumes of plasma for 5 min at 370C; continued generation of CF was then stopped by incubation for 30 min at 56-C. The CF suspensions were diluted with HBSS for the assays. Control suspensions containing no CF were studied in each experiment and included HBSS, bacteria, and immune complexes alone without plasma. Suspensions of bacterial CF were sterilized by passing them through 0.25-jum membrane filters (Millipore Corp., Bedford, Mass.). In the standard assay, equal volumes of plasma and stimulant suspensions were mixed and then diluted to a final plasma (or CF) concentration of 13.3%. For extended chemotaxis studies, final plasma concentrations were varied from 0.1 to 90%. Final plasma concentrations less than 50% (range, 0.1 to 50%) were obtained by diluting suspensions prepared isovolumetrically, as in the standard assay; the maximum concentration of reacted plasma with this method was 50%. Concentrations greater than 50% (range, 10 to 381

382

INFECT. IMMUN.

CASCIATO, GOLDBERG, AND BLUESTONE

90%) were obtained by reacting constant volumes of bacteria or immune complex suspensions with varying volumes of plasma. To insure that these two dilution methods were comparable, concentrations in the 10 to 50% range were included in both methods of dilution. Both concentration ranges were studied simultaneously in each experiment. Measurement of chemotaxis. Chemotaxis was studied by a previously described modification (14) of the Boyden assay (2). The PMN suspension was placed in the upper compartment of a Sykes-Moore tissue culture chamber and separated from the CF suspensions, which were placed in the lower compartment of the chamber, by two Millipore filters. The upper and lower filters had pore sizes of 3 and 0.45 pm, respectively. After an incubation period of 3 h at 370C, the filters were removed and stained. The number of PMNs which had reached the lowermost portion of the 3-pm filter was determined microscopically by counting the cells in 10 high-power fields (HPF). The number of PMNs which had migrated through the 3pm filter and had fallen off were measured by counting the same number of HPF on the uppermost portion of the 0.45-pum filter. All studies were performed in duplicate. The slides were coded and read in a doubleblind fashion. Measurement of random tube migration. Random leukocyte mobility was measured by a modification (3) of the method of Ketchel and Favour (9). For this assay PMNs were obtained from the buffy coat of heparinized blood. Washed plasma-free suspensions of 5 x 10r PMN/ml in 0.1% human albumin were placed in siliconized, microhematocrit tubes and incubated at 370C for 3 h. The distance migrated upward by the leading cells was measured with an ocular micrometer mounted on a microscope, the stage of which was turned to the vertical position. The tubes were placed in a chamber which was constructed from two glass slides and filled with immersion oil (13). The tubes were closed at one end with clay and at the other end with Critocaps (Scientific Products Div., McGaw Park, Evanston, Ill.) to ensure that the assay was performed in the intended atmosphere. All tests were done five times. Anaerobic conditions. Studies on each subject were performed simultaneously under aerobic and anaerobic conditions. An anaerobic glove box (1) containing 85% N2, 10% H2, and 5% CO2 was used for experiments in the absence of oxygen. Suspensions of PMNs and CF were placed into the anaerobic glove box, loaded into the chemotaxis chambers of microhematocrit tubes, and then put into a GasPak jar, which was sealed to maintain the anaerobic conditions. The jar was then removed from the glove box and incubated at 370C. In a parallel set of experiments, the incubation of aerobic chemotaxis chambers either inside or outside GasPak jars had no effect on the results. Entering the anaerobic glove box involved evacuating the air in an exchange box and flushing with anoxic gases several times. To assess the effect of these severe pressure changes on PMNs, a third set of PMN and CF suspensions underwent the pressure changes in the exchange box but were loaded into the chambers and processed in room air. The maintenance of anaerobic conditions in the jars

was monitored with the oxidation-reduction dye resazurin, assuring a redox potential of below -42 mV Eh (15). However, other investigators have demonstrated that the glove box with the gas admixture indicated above contains less than 10 p1 of oxygen per liter and maintains a redox potential of less than -300 mV (11). Additionally, the glove box used in these experiments has proved suitable for the growth of the most fastidious of anaerobic bacteria. Data analysis. Differences in locomotion in the presence and absence of oxygen were evaluated by calculating paired-t statistics. P values greater than 0.01 were defined to be not significant.

RESULTS

Random tube migration. Random capillary tube migration was studied in five subjects. Under aerobic conditions PMNs migrated 5.5 ± 0.2 mm (mean ± standard error), and under anaerobic conditions PMNs migrated 5.1 ± 0.1 mm. There was no significant difference between these results. Chemotaxis. Figure 1 represents the results of chemotaxis assays in 14 subjects under aerobic and anaerobic conditions. All subjects studied are represented except one who demonstrated deficient chemotaxis in response to two types of CF in the presence of oxygen. The values recorded are the numbers of PMNs reaching the lowermost portion of the 3-pm filter. When immune complexes were used, the chemotactic responses in the presence and absence of oxygen S. AUREUS

E.COLI

BFRAGILIS

IMMUNE COMPLEXES

0

280k

0;. 240

4

o

* 0

0

200 *

*

*

A.

ta Aaa

I

60

120

FIG. A. Chem o

FI.1

hmtxsudraerobic anaerobiccodtns andiios;A

,o-

trol suspensions for both conditions (whole bacteria complexes in HBSS without plasma).

or immune

VOL. 21, 1978

CHEMOTAXIS

appeared to be the same. However, when each of the three organimns was used, there was a consistent, markedly diminished chemotactic response under anaerobic conditions. The statistical representation of the chemotaxis results is shown in Table 1. The mean number of PMNs per 10 HPF averaged about 240 with oxygen and only about 50 in the absence of oxygen for each of the three bacteria. On the other hand, approximately 200 PMNs migrated across the filter both with and without oxygen for CF generated from plasma with albumin/antialbumin complexes. Calculating paired-t statistics showed that these differences were highly ificant for each of the bacteria (P < 0.001) and not significant for immune complexes.

None of the control suspensions (stimulants suspended in HBSS without plasma) had chemotactic activity (Fig. 1); albumin-antialbumin complexes and the three bacteria required plasma factors to generate this activity. In a parallel set of experiments, heat inactivation of plasma

at 560C before incubation with bacteria

complexes resulted in the inability of these substances to generate CF activity. When PMNs were exposed to the severe pressure changes involved in entering the anaerobic glove box and then studied for response in aerobic conditions, these PMNs migrated just as well as cells which were processed completely in air. Also, the presence or absence of the bacteria in the CF suspensions did not affect the results. Removing the organdiss from the CF suspensions by Millipore filtration (after the 35-min period of CF generation) had no effect on chemotaxis results. In separate experiments PMNs were counted both on the lowermost surface of the 3-pm filter and on the uppermost surface of the 0.45-pn filter. There was no evidence that the PMNs fell off the upper filter onto the lower filter to any greater or lesser extent under aerobic or anaerobic conditions. Generally, the number of PMNs falling onto the lower filter was proportional to or immune

TABLE 1. Chemotaxis in 14 human subjects PMN/10 HPF under the CF source

following conditions: Aerobic

S.

254

aureus

8b

E. coli

244±6

B. frwgilis

230

Immune

complexes

8

1992

ap

52 + 8

Polymorphonuclear neutrophil chemotaxis under aerobic and anaerobic conditions.

INCT ON AND IMMuNITY, Aug. 1978, p. 381-386 0019-9567/78/0021-0381$02.00/0 Copyright i 1978 American Society for Microbiology Vol. 21, No.2 Printed...
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