CL,N, Table 1 shows the fraction of the added CFU phagocytosed during the incubation period. Cells in both schemes I and II received a single exposure to bacteria but those of scheme II ingested significantly fewer (P < 0.001) bacteria. Scheme III cells which received a double exposure to bacteria had poorer phagocytic capability than did those of scheme I (P < 0.001) but not than did scheme II cells. Since scheme II cells had only a single delayed exposure to bacteria, these data suggest that the reduced phagocytic capability of cells in schemes II and III was probably a consequence of the experimental conditions (centrifugations. etc.) and length of time before exposure of the PMN to bacteria. Thus, there was no significant difference in the phagocytic capability of PMN cells exposed to sequential bacterial stimulation (scheme III) when compared with similarly handled PMN exposed to bacteria only one time (scheme II). Bactericidal Capabilit> Table 2 summarizes the bactericidal capabilities of cells in the various experimental schemes, as expressed in the three ways defined under Materials and Methods. Total remaining bacteria (at 90 min, as fraction of CFU added) was significantly greater following a second finite exposure to bacteria (scheme III) when compared to results of scheme II in which cells were similarly handled but exposed to bacteria a single time (P < 0.02). The smaller number of bacteria remaining in scheme I tubes when compared with scheme III (P < 0.005) may also be a function of differences in bactericidal function consequent to the second bacterial exposure (since the difference between schemes I and II was not statistically significant).

Fraction phagocytosed

of added bacteria during incubation ___~~-... 0.74 (2 O.OlY 0.66 (2 0.02) 0.64 (k 0.03)

Experimental scheme .I II III

periodb .-

-

I vs II 1 vs III I1 vs III ” SEM. b Calculated from ( Between means, ” Not significant.

S. by Student’s

Statistical

significance’

/f 1 = 3.79; ItI = 3.83: n.s.d

P < 0.001 P < 0.001

t test: df >22 in all. P < 0.05 is the level

of significance

used.

PHAGOCYTE

TABLE BACTERICIDAL

Experimental

FUNCTION

129

FATIGUE

2

OF BLOOD LEUKOCYTES FOLLOWING SEQUENTIAL ASSESSMENT IN THREE PARAMETERS

Fraction of added bacteria present 90 min after leukocyte-bacteria incubation represented by

scheme

Total remaining

I II III

0.38 (k 0.03) 0.43 (k 0.02) 0.52 (2 0.02)

Leukocyte-associated

BACTERIAL

EXPOSURE:

30-min change in fraction of added bacteria which was leukocyte associated

0.18 (k 0.02) 0.12 (k 0.02) 0.29 (+ 0.03)

-0.14” -0.12 +0.04

(2 0.04) (k 0.04) (” 0.03)

Statistical significanceb I vs II I vs III II vs III

yip”= 3.92: P < 0.005 ItI = 2.87: P < 0.020

ii;'= 3.OO;P < 0.020 ItI = 3.68; P < 0.005

;;i: t

::;c,:;;

2 8:;;;

0 Minus indicates decrease in fraction during interval. * df = 10 in all tests. c Not significant.

Qualitatively similar results were obtained when PMN bactericidal capability was expressed as the fraction of added bacteria which were detected cell associated (Table 2), in that PMN twice exposed to bacteria showed impaired bactericidal activity compared with similarly handled but only single exposed cells (P < 0.005). Since the course of intracellular killing following a “point” exposure of PMN to bacteria (here, 5 min) will not be confused by continuing ingestion of bacteria, the rate of change in intracellular viable bacteria was also determined (Table 2). Thus, in a 30-min period following single or double exposure to bacteria (schemes II and III, respectively), statistically significant different rates of killing were observed when expressed as the change over a 30-min period in the fraction of added bacteria which was leukocyte associated. Following the second bacterial exposure no detectable change in numbers of intracellular bacteria occurred in 30 min at the time it was studied, while these numbers clearly decreased in cells similarly handled but subjected to only a single exposure to bacteria (P < 0.05). DISCUSSION

The design of these experiments allowed independent assessment of the processes of phagocytosis and bacterial killing by quantitating cell-associated and cellindependent bacteria after a finite period during which a known number of bacteria and PMN were mixed. When human blood phagocytes were subjected to two sequential periods of exposure to S. aureus under the conditions described, their ingestive capacity showed no significant differences from that of cells similarly handled but exposed to bacteria only a single time. When similarly compared, however, a statistically significant impairment in their ability to kill ingested bacteria following the second exposure to bacteria was revealed. It cannot be concluded that no ingestive impairment was present, since some deterioration in that function occurred as a consequence of the experimental conditions required by

130

MATHEISZ

AND

ALLEN

the experiments. Further, it is possible that challenge with even greater numbers of bacteria, or for more than two time intervals, might reveal an ingestive defect. It does seem clear from these data, however, that the killing function of human PMN can be readily fatigued by exposure to bacteria and at a magnitude significantly greater than fatigue (if any) of their ingestive capability. It should be stressed that these studies have involved a repeat exposure of PMN to the same organisms and have studied only the S. aureus. Accordingly, the applicability of our observations to other bacterial species and the specificity of the fatiguing stimulus cannot be determined from the data. Only a limited number of publications have considered the question of efficiency of phagocytic cell activity. In 1940, Hanks (12) explored the effect of increasing numbers of tubercle bacilli on the phagocytic capability of rabbit leukocytes. He found that 75% of the total bacilli were phagocytosed at a bacteria to leukocyte ratio of 0.4: 1 whereas 32.5% were phagocytosed at a ratio of 12: 1. The actual number of bacilli ingested, however, increased from 0.3 bacilli per leukocyte to 3.9. More recently, Clawson er al. (13) in similar studies employed ratios of bacteria to neutrophil of between 1.25 and 4OO:l. At a challenge of 100: 1 capacities for ingestion and degranulation by normal neutrophils reached saturation and did not decline. The percentage of the total bacteria present which was killed in 60 min declined from 79% at a ratio of 1.4: 1 to 47% at 103: 1. The number of bacteria killed per neutrophil, however, again increased from 1.1 to 47.7 for the ratios 1.4: 1 and 103: 1 respectively. Thus, when looked at from the point of view of cellular activity, both studies suggest that the more bacteria present in the environment, the more bacteria can be ingested (to a limit) or killed by each phagocytic cell. Our experiments have tested the effect on PMN of a second exposure to bacteria within similar bacteria per neutrophil ratios and not simply as a function of increasing ratios. Under these conditions diminished bactericidal capacity occurs, suggesting that duration of exposure rather than just numbers of bacteria in the environment is the most important determinant in this functional fatigue. We have performed a limited number of experiments utilizing lower bacteria:PMN ratios which have failed to reveal this phenomenon. Either a threshold density of exposure is required before bactericidal fatigue develops with time, or the magnitude of fatigue is directly related to the density of exposure. Minor degrees of fatigue could be missed by the limits of discrimination of the techniques used. These experiments offer no clue as to the mechanism responsible for deterioration of PMN bactericidal capability, though depletion of some intracellular component necessary for intracellular killing offers the most appealing hypothesis. Whether the fatigue is specifically related to this test organism, could be induced for one species by exposure to another or could be caused by ingestion of inert particles have not been determined. Reduced functional capability of human neutrophils during severe bacterial infections has been previously noted (5-10). From the data presented here it appears that prolonged bacterial stimulation of neutrophils may be responsible for decreased bactericidal activity. This interpretation offers an explanation for Solberg’s finding (7) that in neutrophils obtained from patients with severe bacterial infections, large numbers of viable bacteria were located intracellularly, suggest-

PHAGOCYTE

FATIGUE

131

ing that ingestion occurred normally but intracellular killing of bacteria was reduced. They also offer a basis for, and support of, Koch’s data (9) which suggest that bacterial ingestion by phagocytic cells is normal or slightly increased during bacterial infection while bactericidal activity is defective, which he attributes to malfunction of myeloperoxidase-mediated bactericidal systems. The results of our experiments suggest that the effectiveness of antibiotic therapy in treatment of bacterial infections may be related in part to its ability to decrease numbers of extracellular bacteria, thus allowing the neutrophil to escape the fatiguing effect of continuing bacterial exposure on its bactericidal capability. ACKNOWLEDGMENTS J.S.M. was supported by an Allied Health Professions Advanced Traineeship from the USPHS.

REFERENCES 1. Holmes, B., Quie, P. G., Windhorst, D. B., and Good, R. A., Lance? 1, 1225, 1966. 2. Holmes, B., Park, B. H., Malawista, S. E., Quie, P. G., Nelson, D. L., and Good, R. A., N. Engl. .I. Med. 283, 217, 1970. 3. Cooper, M. R., DeChatelet, L. R., McCall, C. E., LaVia, M. F., Spur-r, C. L.. and Baehner, R. L., J. Clin. Invest. 51, 769, 1972. 4. Gray, G. R., Stamatoyannopoulos, G., Naiman, S. C., Kliman, M. R., Klebanoff, S. J.. Austin, T., Yoshida, A., and Robinsin, G. C. F., Lancer 2, 530, 1973. 5. Copeland, J. L., Karrh, L. R., McCoy, J., and Guckian, J. C., Texas