Effect of Methylprednisolone on Bacterial Clearance and Endotoxin Liberation during Experimental Sepsis Induced by Gram-Negative Bacteria
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INFECTION AND IMMUNITY, Apr. 1986, p. 26-30 Vol. 52, No. 1
0019-9567/86/040026-05$02.00/0
Copyright C 1986, American Society for Microbiology
Effect of Methylprednisolone on Bacterial Clearance and Endotoxin
Liberation during Experimental Sepsis Induced by Gram-Negative
Bacteria
PATRICIA M. FLYNN, JERRY L. SHENEP,* DENNIS C. STOKES, WILLIAM K. HILDNER, PAUL W.
MACKERT, RICHARD L. SNELLGROVE, AND JEROLD E. REHG
Divisions of Infectious Diseases, Cardiopulmonary Diseases, and Comparative Medicine, St. Jude Children's Research
Hospital, Memphis, Tennessee 38101, and Department of Pediatrics, University of Tennessee Center for the Health
Sciences, Memphis, Tennessee 38104
Received 21 November 1985/Accepted 18 December 1985
To determine the effect of methylprednisolone administration on the clearance of bacteremia and the release
and clearance of endotoxin during antibiotic therapy of gram-negative bacterial sepsis, Escherichia coli Kl
sepsis was induced in paired rabbits. Moxalactam and either methylprednisolone or placebo were administered
to infected rabbits 1.5 h after intraperitoneal administration of live bacteria. Serial blood samples were
obtained for quantitation of bacteremia and endotoxemia, arterial blood gases, and complete blood count.
Arterial blood pressure, heart rate, and core body temperature were also monitored. There were no significant
differences between the methylprednisolone-treated and placebo-treated groups in either the levels of
bacteremia or endotoxemia or in the physiologic, metabolic, or hematologic parameters that were -measured.
We conclude that methylprednisolone administration has no acute effect on bacterial clearance or on the
kinetics of endotoxin release and clearance during antibiotic therapy of gram-negative bacterial sepsis in this
experimental model.
Although the role of corticosteroid administration in the agar. Two grams of porcine mucin (Sigma Chemical Co., St.
therapy of gram-negative bacterial sepsis has been exten- Louis, Mo.) was added with vigorous stirring to 40 ml of the
sively investigated in experimental animal models and in overnight culture, and the resulting suspension was held on
clinical settings (2, 10, 11, 15), little is known about the effect ice until used.
of corticosteroid administration on the clearance of bactere- Experimental infection of rabbits. Twelve New Zealand
mia and endotoxemia. Corticosteroid administration is White rabbits from a local rabbitry, weighing 2.7 to 3.9 kg,
known to decrease neutrophil phagocytosis (4) and to received antibiotic-free food and water ad libitum. Paired
acutely depress the ability of the lungs to clear gram- rabbits differed in weight by no more than 0.3 kg (VOL. 52, 1986 METHYLPREDNISOLONE AND BACTERIAL. CLEARANCE 27
107 A A B105C
105 ~~~~~~B
0C
06 04
J0j
TI~~~~~~~~~~~~~~~~1
io5~~~~~~~~~~~~~~~~~~~~~~~7
04 10""12 I,~~~~~~C
102-
LL-
103 10 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
l102
10~ ~ ~ ' 1.I0 '10
Time (hrs) Time ( hrs) Time (hrs)
FIG. 1. Geometric mean levels of bacteremia (A), plasma free endotoxin (B), and plasma total endotoxin (C) for the methylprednisolone
(O) and placebo (0) treatment groups. Animals received E. coli intraperitoneally at 0 h and treatment with moxalactam and either
methylprednisolone or placebo at 1.5 h. Each bar represents one standard deviation of the mean of six paired trials.
catheter was flushed with 10 ml of 0.9% sodium chloride to that of the plasma sample. The validity and specificity of
after each' sampling period. this assay for endotoxin has been previously confirmed (13).
After obtaining the blood sample at 1.5 h, each rabbit Physiologic measurements. Mean arterial pressure, core
received moxalactam (100 mg/kg; Eli Lilly & Co., Indianap- body temperature, and heart rate were continuously moni-
olis, Ind.) as an intra-arterial bolus. By random selection, tored (Seimens 404), and recorded on a strip chart recorder
one of the paired rabbits also received methylprednisolone (Western Graphtec).
sodium succinate (30 mg/kg; Abbott Laboratories, North Statistical analysis. Student's t test was used to perform
Chicago, Ill.), and the other received an equal volume of statistical analysis of data with a model HP41-CV program-
0.9% sodium chloride as an intra-arterial bolus infusion. All mable calculator (Hewlett-Packard Co., Corvalis, Oreg.).
laboratory and physiologic measurements were performed in Significance was determined at P < 0.05.
a blind fashion by an investigator. All surviving rabbits were
sacrificed 7.5 h after infection. RESULTS
Quantitation of bacteremia. Serial 10-fold dilutions of each Effect of methylprednisolone on bacteremia. Before the
blood sample were prepared with phosphate-buffered saline administration of moxalactam, the geometric mean bacterial
containing 1% albumin (pH 7); 0.1 ml of each dilution was counts were 8.5 x 103 and 3.1 x 104 CFU/ml of blood for the
cultured on tryptic soy agar, and CFU were counted after methylprednisolone and placebo treatment groups, respec-
overnight incubation at 37°C. tively (Fig. 1A). The geometric mean levels of bacteremia
Endotoxin assay. Free endotoxin was separated from bac- peaked 1 h after moxalactam administration (2.5 h after
terial cell-bound endotoxin by filtration as previously de- infection) at 5.9 x 104 and 3.2 x 104 CFU/ml of blood for the
scribed (13). Levels were quantitated with the Pyrotell methylprednisolone and placebo treatment groups, respec-
Limulus lysate gelation assay (Associates of Cape Cod, tively. The geometric mean levels of bacteremia declined
Woods Hole, Mass.). Plasma inhibitors of the Limulus assay throughout the remaining observation period in both groups.
were inactivated by diluting the plasma sample 1:3 with Although the methylprednisolone-treated group exhibited a
pyrogen-free water and heating to 100°C for 10 min. Serial trend toward higher levels of bacteremia, at no time were the
twofold dilutions of plasma samples were assayed in parallel mean levels of bacteremia in the two groups significantly
with known concentrations of reference E. coli endotoxin different. Five animals, three in the placebo-treated group
(Associates of Cape Cod). Results were expressed as the and two in the methylprednisolone-treated group, com-
amount of U.S. standard endotoxin with activity equivalent pletely cleared bacteremia.28 FLYNN ET AL. INFECT. IMMUN.
Effect of methylprednisolone on endotoxemia. Geometric
mean levels of total and free endotoxin were similar in both 140
treatment groups before antibiotic administration. After an-
tibiotic administration and concomitant with declining levels
of bacteremia, levels of plasma total and free endotoxin 120
increased rapidly (Fig. lB and C). The levels appeared to j -
peak 6 h after antibiotic administration. At no time were the
plasma levels of either total or free endotoxin significantly - 100
different for the methylprednisolone and placebo treatment 0
groups (P > 0.05, each sample time). of 80
Effect of methylprednisolone on the physiologic, metabolic,
and hematologic status of septic rabbits. Just before infection 60
the mean arterial pressure in the placebo treatment group
was 70 torr compared with 66 torr in the methylprednisolone
treatment group (Fig. 2). At the time of antibiotic adminis- 40
tration and either placebo or methylprednisolone adminis- 60
tration, the mean arterial pressure had decreased to 51 and
42 torr, respectively. The blood pressure in both groups 0
continued to decline, stabilizing only near the end of the 2 40
observation period. At no time was the difference between cm
the treatment groups statistically significant (P > 0.05, each " 20
sample time). Neither mean heart rates or mean core body
temperatures were significantly different between the two
treatment groups (P > 0.05, each sample time; Fig. 2). 0 . .
Arterial blood gas findings are illustrated in Fig. 3. After J 40
infection the mean PaO2 rose initially as the animals ,,
E
20
z0O
80 0
0 2 4 6 8
~~~~T
~~~~2
60
~~~~~~~~~~~~~~~' ime (hrs)
-¾<
60 | FIG. 3. Arterial blood gas determinations for methylpredniso-
° 40 llone (0) and placebo (0) treatment groups. Each bar represents one
2 o l standard deviation of the mean of six paired trials.
20
hyperventilated in response to their metabolic acidosis and
180 then remained stable throughout the remainder of the obser-
vation period. Although PaO2 levels in the methylpredniso-
160 I lone treatment group were lower than those in the placebo
. treatment group after corticosteroid therapy, at no time was
_ 10 ~ .the difference significant (P > 0.05 each sample time). Serial
.'140 p ^, , PaCO2 and serum bicarbonate concentrations were similar
- ' , forw both treatment groups (Fig. 3) throughout the observa-
,, 120 L T 1'/l T B < I tion period, demonstrating progressive metabolic acidosis
cr_
IQ T P/ | with respiratory compensation. No significant difference was
T I % noted between the two treatment groups (P > 0.05, each
< 100 li sample time).
Sequential measurements of total leukocyte, platelet and
hemoglobin concentrations are presented in Table 1. Con-
80 centrations of leukocytes and platelets declined markedly
after infection in both treatment groups, whereas hemoglo-
60 bin concentrations were relatively stable. There was no
r______ significant difference between the two treatment groups for
40 any of these parameters (P > 0.05, each sample time).
Y
U2; 30°. X j( 4 > In the methylprednisolone-treated group, two rabbits ex-
pired 6.5 h after infection and one rabbit expired 7.5 h after
1 .__.__.__.__.__,__.__|infection. In the placebo-treated group, two rabbits expired
0 2 4 6 8 6.5 h after infection.
Time (hrs) DISCUSSION
FIG. 2. Mean arterial blood pressure, heart rate, and core body The administration of corticosteroids in conjunction with
temperature for the methylprednisolone (0) and placebo (0) treat- antibiotics has clearly reduced mortality rates in some ex-
ment groups. Each bar represents one standard deviation of the perimental models of gram-negative bacterial sepsis (2, 5, 6,
mean of six paired trials. 10). Although the mechanism of this protection is not under-VOL. 52, 1986 METHYLPREDNISOLONE AND BACTERIAL CLEARANCE 29
TABLE 1. Effect of methylprednisolone administration on the model. However, after infection, bacteria present in blood
mean blood levels of leukocytes, platelets, and hemoglobin during are lysed by the action of antibiotics, liberating large
antibiotic therapy of experimental E. coli sepsis" amounts of endotoxin (13). This relation between bacterial
h after Results with placebo group/methylprednisolone group
lysis and endotoxin liberation closely parallels observations
bacterial 103 Hemoglobin of bacterial clearance and endotoxin liberation in patients
challenge Leukocytes/Il Platelets/t.l (g/100 ml) receiving antibiotics for gram-negative bacterial sepsis (J. L.
Shenep, F. F. Barrett, G. L. Stidham, D. F. Westen-
0 4,700/3,800 303/329 11.6/11.4 kirchner, and P. Flynn, Crit. Care Med. 13:298, 1985).
1.5b 1,800c/2,300c 265/315 11.9/11.9 Although corticosteroids clearly mitigate some of the toxic
3.5 1,060c/730c 149/7c 12.2/11.0
effects of endotoxin, their impact in the therapy of gram-
5.5 1,000c/850c 38c/19c 9.4c/10.4
negative bacterial sepsis is less clear. Despite the fact that
a None of the differences between treatment groups is statistically signifi- bacterial clearance and endotoxin liberation are readily
cant. influenced in this model (12), the results of the current study
b Methylprednisolone or
placebo was administered with moxalactam.
c Significantly different from the mean at time 0 as assessed by the did not demonstrate an adverse effect of methylprednisolone
comparison of two means t test. administration on bacterial clearance during antibiotic ther-
apy for gram-negative bacterial sepsis. On the other hand,
this study revealed no beneficial effect of methylpredniso-
stood, proposed explanations include an intrinsic anti- lone administration on either the level of endotoxin or on
endotoxin activity of corticosteroids (1), the prevention of endotoxin-sensitive physiologic, metabolic, or hematologic
endotoxin-induced hypoglycemia (5), improved circulation parameters in the present model. To date, clinical studies of
and distribution of antibiotics (5), and the prevention of the effectiveness of corticosteroid administration in prevent-
complement-induced granulocyte aggregation (3). Enhanced ing mortality in gram-negative bacterial sepsis are collec-
clearance of bacteremia or endotoxemia could also be a tively inconclusive. However, in one distinct subset of
beneficial factor, but these potential effects have been the patients, those with typhoid fever, a clear benefit appears to
subject of few investigations. On the other hand, as result from corticosteroid administration (7). Further
corticosteroids are known to decrease neutrophil phagocytic progress in understanding both the pharmacologic and
function (4) and contribute to superinfection (15), corticoste- microbiologic effects of corticosteroid administration during
roid administration could potentially result in decreased therapy of gram-negative bacterial sepsis may eventually
clearance of bacteremia. Consistent with this idea Skornik permit delineation of other subsets of patients with gram-
and Dressler reported that administration of corticosteroids negative bacterial infections that may benefit from cortico-
to both normal and burned rats acutely depressed clearance steroid administration.
of Pseudomonas aeruginosa from the lung (14). In the
present model of gram-negative bacterial sepsis, levels of ACKNOWLEDGMENTS
bacteremia were not significantly different between the This work was supported by Public Health Service grant
methylprednisolone and placebo treatment groups. Rather, RR-00584-20 from the Division of Research Resources, National
levels of bacteremia appeared to be influenced solely by Institutes of Health, by grant CF-6040 from the American Cancer
antibiotic administration (Fig. 1A). Society, and by the American-Lebanese-Syrian Associated Chari-
In a recent report, Johnston and Greisman (8) examined ties.
the effect of corticosteroid administration on endotoxin LITERATURE CITED
levels in a murine model of gram-negative bacterial sepsis.
Mice that were treated with kanamycin alone had similar 1. Greisman, S. E. 1982. Experimental gram-negative bacterial
plasma endotoxin levels but a higher mortality rate when sepsis: optimal methylprednisolone requirements for prevention
compared with mice that received kanamycin and of mortality not preventable by antibiotics alone. Proc. Soc.
Exp. Biol. Med. 170:436-442.
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sent data show no effect of methylprednisolone administra- Experimental gram-negative bacterial sepsis: prevention of
tion on the plasma levels of either total or free endotoxin. mortality not preventable by antibiotics alone. Infect. Immun.
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