INFECTION

AND

IMMUNITY, Aug. 1976,

Vol. 14, No. 2 Printed in U.S.A.

p. 399-402

Copyright C 1976 American Society for Microbiology

Granulocyte Function and Pseudomonas Burn Wound Infection D. D. McEUEN, G. C. GERBER, P. BLAIR, AND K. EURENIUS* Departments ofPathology and Medicine,* Medical University of South Carolina, Charleston, South Carolina 29401

Received for publication 18 March 1976

Granulocyte function in burn injury has been evaluated with an in vitro animal assay. Bactericidal activity, using this technique, was markedly depressed only when granulocytes from scald burned rats were challenged with each of three strains ofPseudomonas aeruginosa in the presence of autologous postburn serum or heat-inactivated serum. Preincubation of postburn and normal serum at 37°C for 1 h resulted in normal phagocytosis. These studies validate an investigation of plasma and/or granulocyte replacement therapy in the burned patient.

Infection with Pseudomonas aeruginosa remains the major cause of death in burn patients. Attempts to eliminate such infection with the use of topical antibacterial therapy and systemic antibiotics have been only minimally effective and point to the necessity of a better understanding of the burn patient's host defense. Although alterations in humoral immune defense mechanisms have been cited (2, 8, 15, 16), the experimental and clinical use of immune vaccines and passive immunization with pseudomonas antibody have not altered mortality in this disease state. Impaired granulocyte function (3), granulocytopenia (17), and altered serum opsonic activity (4) have all been implicated as crucial defects in host defense in the presence of a pseudomonas infection. Recently, we have quantitated the relationship between normal granulocyte function and mortality in an infected, burned rat model by using three distinct strains of P. aeruginosa (14). Using similar techniques, we have now measured in vitro bactericidal activity in these same organisms by granulocytes from scald burned rats. Additionally, attempts were made to distinguish between cellular and serum-mediated abnormalities, since such data have significant bearing upon the efficacy of granulocyte replacement therapy. MATERIALS AND METHODS In vitro granulocyte bactericidal activity upon three strains of P. aeruginosa was measured after infliction of a 30% third-degree scald burn (21). (These three strains were donated by Robert B. Lindberg at the U.S. Army Institute of Surgical Research, Fort Sam Houston, Tex. In our studies, these are referred to as strains I, II, and III, respectively, and have been shown to induce distinct mor-

tality rates in the scald burned rat.) At 4 h and 1, 2, 3, 5, and 10 days postburn, intraperitoneal granulocytes were collected by a modification of the method of Hirsch (13). Cells were adjusted to a final concentration of 5 x 106/ml. Three strains ofP. aeruginosa, subcultured and stored at -70°C, were each seeded in Trypticase soy broth at 37°C. After 18 h of growth, final bacterial concentrations of 5 x 106, 109, and 1010 bacteria/ml ofculture were prepared for strains I, II, and III, respectively. Such concentrations, when incubated with 5 x 106 granulocytes from normal unburned controls, resulted in 90% bactericidal activity, Assays were performed as a modification of the technique of Sbarra et al. (19). Experimental tubes included granulocytes, bacteria, autologous serum (40%), and Krebs-Ringer phosphate medium. Control tubes were similarly constituted without leukocytes. After a 90-min incubation, samples of both control and experimental reactions were removed, serially diluted, and plated in Trypticase soy agar. After incubation at 37°C for 24 h, bacterial colonies were counted and corrected for dilutions, and comparisons were made between control and experimental groups.

RESULTS Leukocyte recovery from animals after bum injury was markedly diminished when compared to control animals (Table 1) and remained low even at 10 days postbum. Differential cell counts of peritoneal exudates are included for both control and 2-day postburn animals and indicate that, whereas total yields were markedly decreased, mature, granulocyte, eosinophil, lymphocyte, and monocyte components of the cell compartment remained constant. When peritoneal granulocytes collected from postbum animals were challenged by each of the three strains of P. aeruginosa, a 399

400

INFECT. IMMUN.

McEUEN ET AL.

marked depression in bactericidal activity was observed at all time periods (Fig. 1). In an attempt to isolate an inhibitor or a deficiency causing this decrease in bactericidal activity, we performed the experiments shown in Fig. 2. Either normal or 2-day postburn granulocytes (5 x 106 cells/ml) were incubated with strain I P. aeruginosa (108 organisms/ml) in the presence of either normal, 2-day postburn, or attenuated serum. In experiments A through D, decreased bactericidal activity occurred only when postburn granulocytes were incubated with postburn serum (experiment TABLE 1. Peritoneal granulocyte recovery Determination Yield

Differential

Time n post-

post

n

Control 4h 1 day 2 days 3 days 5 days 10 days

18 11 10 12 10 11 10

Control

20 8

2 days

recov-% 10-7) graul+

Mature

Cel r

SEM

18.8 2.1 1.1 1.6 2.8 3.1 3.5

± 2.09 ± 1.3 ± 0.9 ± 0.3 ± 0.6 ± 0.8 ± 0.7

ery (x

cytes±-

85.9 ± 2.3 81.2 ± 6.3

SEM

3 DAYS

5

DAYS

10 DMS

TIME POSTBURN

FIG. 1. Percent bactericidal activity of P. aerugistrains I (0), II (0), and III (U); 5 x 106 P. aeruginosa organisms were incubated with 5 x 10" granulocytes in the presence of 40% autologous postburn serum. Studies were performed at 4 h and 1, 2, 3, 5, and 10 days postburn. Bactericidal activity is expressed as percentage of control (mean values + 1 standard error of the mean). nosa

CELLS

N

N

B

B

8

B

B

B

B

n.

6

6

12

12

6

6

3

3

6

iO

>

+ ni

(a + N37*.6)

-

ri,

+

w E 40 _j

04 e

cc

W

°+,

4t

202

.-4 e

c

E

F

G

H

FIG. 2. Percent bactericidal activity of P. aerugistrain I by granulocytes from normal and burned rats. Nine assays (A-I) were performed using combinations of granulocytes and 40% serum from both burned and normal donors: A, normal cells and normal serum; B, normal cells and postburn serum; C, postburn cells and normal serum; D, postburn cells and postburn serum; E, postburn cells and an equal mixture of postburn and normal serum; F, postburn cells and an equal mixture ofpostburn and normal serum preincubated at 37°C for 60 min; G, postburn cells and buffer (Krebs-Ringer phosphate medium); H, postburn cells and heat-inactivated normal serum (55°C, 20 min); I, postburn cells and an equal combination of postburn serum and heatinactivated normal serum. n, Number of assays performed. Mean values are presented + 95% confidence limits of the mean. *P < 0.01 when compared to experiment C. **P < 0.001 when compared to experiment C. nosa

I

2 DAYS

B

PCO.OOI WHEN COMPARED WITH C

n9

DAY

N

*P< 0.01 WHEN COMPAED WITH C

'i

4 HOURS

B

EXPERIMENT NO.

i4

w

N

A

a Total leukocyte recovery from rat peritoneal fluid 18 h after sodium caseiviate infusion. Granulocyte concentrations of these leukocyte populations are presented for both control and 2-day postburn animals. n, Number of animals studied; SEM, standard error of the mean.

NORMAL RESPONSE

(204 -552)

SERUM

D). It should be noted that both normal granulocytes (experiment B) and postburn granulocytes (experiment C) had normal bactericidal activity in the presence of normal serum. The addition to postbum granulocytes of a mixture of burn and normal sera either immediately (experiment E) or after 1 h of preincubation at 3700 (experiment F) also resulted in normal bactericidal activity. When normal serum was used but first heated (55°0, 20 min), bactericidal activity was markedly diminished (experiment H), even below the level seen with postburn serum (experiment D). Finally, the combination of postburn serum plus heat-treated normal serum was incapable of correcting the defect (experiment I).

VOL. 14, 1976

GRANULOCYTES AND PSEUDOMONAS

DISCUSSION Impaired granulocyte function has been implicated in the pathogenesis of infections complicating a number of clinical disorders. Defective intracellular killing of phagocytosed bacteria has been reported in patients with Chronic Granulomatous Disease of Childhood (18), Chediak-Higashi syndrome (10), and myeloproliferative disorders (11). Moreover, patients with diabetes mellitus (6) and lymphoproliferative diseases (19) have demonstrated depressed granulocyte phagocytosis. P. aeruginosa has low invasiveness in healthy individuals but can cause sepsis in patients who have serious underlying diseases. The increased incidence of pseudomonas infection in acute leukemia has been correlated with absolute granulocytopenia (20). We have previously reported (7) early depletion of bone marrow granulocyte stores in scald burned rats and concomitant depletion ofthe marginating granulocyte compartment. This absolute granulocytopenia was magnified in the presence of an experimental pseudomonas infection of the burn wound (17). Whereas pseudomonas is not invasive in the normal rat, it is lethal in the scald burned animal. Recent investigations with three strains of Pseudomonas correlate the mortality produced by infection with resistance to bactericidal activity by normal granulocytes (14). We suggest that suppression of host defense in burns may also be directly related to granulocyte and serum abnormalities. The current studies demonstrate depressed bactericidal activity by postburn granulocytes in the presence of autologous postburn serum or heat-labile normal serum. It would appear that granulocytes from bumed animals lack an essential factor(s) for the phagocytosis of P. aeruginosa and that this defect can be corrected by incubation with normal serum but not by incubation with autologous postburn serum. The necessary factor(s) in normal serum is heat labile. Studies were not performed to identify this material(s) or to determine whether an identical defect is present on bum cells. However, these studies are worthy of consideration. It may be, for example, that a number of defects in bum cells and burn serum may exist and that normal cells phagocytose in the presence of bum serum because such cells have been exposed to this material in vivo. Since neither burned nor normal granulocytes were capable of phagocytosis of pseudomonas in the absence of either serum, it is clear that other serum factors must play a role in this process. In the absence of granulocytes, bacterial cell growth measured by colony counts was constant in the

presence of either buffer or normal, burned, or attenuated serum. Alexander (1) has reported both a deficiency in lysosomal enzymes and decreased intracellular killing of bacteria by granulocytes from burned patients in the presence of normal pooled serum. We were unable to identify a suppression in bactericidal activity in granulocytes from burned animals unless these cells were incubated in autologous postburn serum. The importance of reactions between granulocytes and serum factors that result in chemotaxis, phagocytosis, and degranulation has been established (5, 12). Recovery of peritoneal granulocytes by casein infusion was markedly reduced in our animals after burn injury. Whereas this could be explained by postburn granulocytopenia (7), it might also be a reflection of altered chemotaxis. Warden et al. (22) have recently demonstrated decreased leukocyte migration in granulocytes from 50 burned patients, using casein as a stimulus. Bjornson and Alexander (4) have reported reduced opsonic activity in the sera of burned patients. This defect could be corrected by mixing burned sera with normal sera but reappeared when the two sera were preincubated. In our studies, no inhibitor could be demonstrated either when burned serum was mixed with normal serum or when burned serum was preincubated with normal serum for 1 h. These in vitro studies demonstrate abnormalities in bactericidal activity that occur only in the coincubation of postburn granulocytes and postburn or heat-treated serum. Fjellstrom and Arturson (9), and Bjornson and Alexander (4) have demonstrated marked suppression of functional complement activity in the sera of burned patients. These observations coincide with serum defect(s) that we have described. We believe these data justify an investigation of the feasibility and efficacy of plasma and/or granulocyte replacement therapy in the burned subject to reduce the incidence and morbidity of pseudomonas infections.

401

ACKNOWLEDGMENT This work was supported by Public Health Service research grant GM21758-01 from the National Institute of General Medical Sciences. LITERATURE CITED 1. Alexander, J. W. 1967. Serum and leukocyte lysosomal enzymes: derangements following severe thermal injury. Arch. Surg. 95:482-491. 2. Alexander, J. W., and J. A. Moncrief. 1966. Alterations of the immune response following thermal injury in the rat. Arch. Surg. 93:75-83. 3. Alexander, J. W., and D. Wixson. 1970. Neutrophil dysfunction and sepsis in burn injury. Surg. Gynecol. Obstet. 130:431-438. 4. Bjornson, A. B., and J. W. Alexander. 1974. Alterations

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of serum opsonins in patients with severe thermal 14. McEuen, D. D., P. Blair, V. C. DelBene, and K. Eureninjury. J. Lab. Clin. Med. 83:372-382. ius. 1976. Correlation between pseudomonas burn 5. Bjornson, A. B., and J. G. Michael. 1973. Factors in wound infection and granulocyte antibacterial activnormal serum that promote bacterial phagocytosis. J. ity. Infect. Immun. 13:1360-1362. Infect. Dis. 128(Suppl):S182-S186. 15. Munster, A. M. 1970. Alterations of the host defense 6. Drachman, R. H., R. K. Root, and W. B. Wood. 1966. mechanisms in burns. Surg. Clin. North Am. Studies on the effect of experimental nonketotic dia50:1217-1225. betes mellitus on antibacterial defense. I. Demon- 16. Munster, A. M., and H. C. Hoagland. 1969. Serum stration of a defect in phagocytosis. J. Exp. Med. immunoglobulin patterns after burns. Surg. Forum 124:227-240. 20:76-77. 7. Eurenius, K., and R. 0. Brouse. 1973. Granulocyte 17. Newsome, T. W., and K. Eurenius. 1973. Suppression of kinetics after thermal injury. Am. J. Clin. Pathol. granulocyte and platelet production by pseudomonas 60:337-342. burn wound infection. Surg. Gynecol. Obstet. 8. Eurenius, K., and R. F. Mortensen. 1971. The phytohe136:375-379. magglutinin (PHA) response in the thermally injured 18. Quie, P. G., J. G. White, B. Holmes, and R. A. Good. rat. Int. Arch. Allergy Appl. Immunol. 40:707-714. 1967. In vitro bactericidal capacity of human polymor9. Fjellstrom, K. E., and G. Arturson. 1963. Changes in phonuclear leukocytes: diminished activity in chronic the human complement system following burn Granulomatous Disease of Childhood. J. Clin. Intrauma. Acta Pathol. 59:257-270. vest. 46:668-679. 10. Gallin, J. I., J. S. Bujak, E. Patten, and S. M. Oliff. 19. Sbarra, A. J., W. Shirley, R. J. Selvaraj, E. Ouchi, and 1974. Granulocyte function in the Chediak-Higashi E. Rosenbaum. 1967. The role of the phagocyte in Syndrome in mice. Blood 43:201-206. host-parasite interactions. I. The phagocytic capabili11. Goldman, J. M., and K. H. Th'ng. 1973. Phagocytic ties of leukocytes from lymphoproliferative disorders. function of leukocytes from patients with acute myeCancer Res. 24:1958-1968. loid and chronic granulocytic leukaemia. Br. J. Hae- 20. Sickles, E. A., V. M. Young, W. H. Greene, et al. 1973. matol. 25:299-308. Pneumonia in acute leukemia. Ann. Intern. Med. 12. Henson, P. M. 1974. Complement-dependent platelet 79:528-534. and polymorphonuclear leukocyte reactions. Trans- 21. Walker, H. L., and A. D. Mason. 1968. A standard plant. Proc. 6:27-31. animal burn. J. Trauma 8:1049-1051. 13. Hirsch, J. G. 1956. Phagocytin: a bactericidal substance 22. Warden, G. D., A. D. Mason, and B. A. Pruitt, Jr. 1974. from polymorphonuclear leukocytes. J. Exp. Med. Evaluation of leukocyte chemotaxis in vitro in ther103:589-611. mally injured patients. J. Clin. Invest. 54:1001-1004.

Granulocyte function and Pseudomonas burn wound infection.

INFECTION AND IMMUNITY, Aug. 1976, Vol. 14, No. 2 Printed in U.S.A. p. 399-402 Copyright C 1976 American Society for Microbiology Granulocyte Fu...
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