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

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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
therapy of gram-negative bacterial sepsis has been exten-
sively investigated in experimental animal models and in
clinical settings (2, 10, 11, 15), little is known about the effect
of corticosteroid administration on the clearance of bactere-
mia and endotoxemia. Corticosteroid administration is
known to decrease neutrophil phagocytosis (4) and to
acutely depress the ability of the lungs to clear gram-
negative bacteria (14). Uptake of lipopolysaccharide by the
liver, spleen, adrenal gland, and lung is also altered in the
presence of corticosteroids (9). Thus, it is reasonable to
postulate that corticosteroid administration may alter the
host's ability to clear bacteremia and endotoxemia during
gram-negative bacterial sepsis.
To investigate the effects of corticosteroid administration
on the clearance of bacteremia and endotoxemia, we studied
paired rabbits with experimentally induced Escherichia coli
Kl sepsis that were treated with an antibiotic plus either
methylprednisolone or placebo. Serial blood samples were
obtained from these rabbits for quantitation of bacteremia
and plasma endotoxin levels. Endotoxin-sensitive physio-
logic, metabolic, and hematologic parameters were also
measured. The results of this study provide additional data
with which to weigh the potential risks and benefits of
corticosteroid administration in the therapy of gram-negative
bacterial sepsis.
MATERIALS AND METHODS
Bacteria. E. coli K1:07 strain C94 was stored in skim milk
at -70°C and recovered on tryptic soy agar. After overnight
incubation at 37°C five representative colonies were
transferred into 50 ml of tryptic soy broth and incubated
overnight at 37°C. The concentration of bacteria was
determined by quantitative dilution and culture on tryptic soy
*
Corresponding author.
26
agar. Two grams of porcine mucin (Sigma Chemical Co., St.
Louis, Mo.) was added with vigorous stirring to 40 ml of the
overnight culture, and the resulting suspension was held on
ice until used.
Experimental infection of rabbits. Twelve New Zealand
White rabbits from a local rabbitry, weighing 2.7 to 3.9 kg,
received antibiotic-free food and water ad libitum. Paired
rabbits differed in weight by no more than 0.3 kg (<10%
difference). Paired rabbits were anesthetized by intramuscu-
lar injection of a 3:3:1 mixture of ketamine (100 mglml),
xylazine (20 mg/ml), and acepromazine (10 mg/ml) at a
dosage of 0.7 ml/kg. This drug preparation contained no
endotoxin as assessed by the assay described below. Anes-
thesia was maintained by intramuscular administration of 0.1
ml/kg of this solution as needed. An indwelling femoral
artery catheter was surgically placed in each rabbit and
perfused with pyrogen-free 0.9% sodium chloride at a rate of
3 ml/h.
The 40-ml suspension containing live bacteria and porcine
mucin was divided into two 20-ml sample, and one sample
was administered intraperitoneally to each of the paired
rabbits. The challenge dose contained from 3.1 x 108 to 3.5
x 109 CFU of E. coli per ml.
Immediately before bacterial challenge and at 1, 1.5, 2,
2.5, 3.5, 4.5, 5.5, 6.5, and 7.5 h after bacterial challenge, 3-ml
blood samples were obtained from each rabbit and divided
equally between two sterile pyrogen-free Falcon tubes
(Becton, Dickinson and Co., Oxnard, Calif.) each containing
0.07 ml of preservative-free heparin (Weddel Pharmaceuti-
cals, Ltd., London) and stored on ice for determinations of
endotoxin. At each time interval 0.5 ml of blood was also
obtained for arterial blood gas determination (model ABL3;
Radiometer). Before bacterial challenge and at 1.5, 3.5, and
5.5 h after challenge, 40 ,ul of blood was obtained for
complete blood count (Cell-Dyne 900). The femoral arterial
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-

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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
after each' sampling period.
After obtaining the blood sample at 1.5 h, each rabbit
received moxalactam (100 mg/kg; Eli Lilly & Co., Indianap-
olis, Ind.) as an intra-arterial bolus. By random selection,
one of the paired rabbits also received methylprednisolone
sodium succinate (30 mg/kg; Abbott Laboratories, North
Chicago, Ill.), and the other received an equal volume of
0.9% sodium chloride as an intra-arterial bolus infusion. All
laboratory and physiologic measurements were performed in
a blind fashion by an investigator. All surviving rabbits were
sacrificed 7.5 h after infection.
Quantitation of bacteremia. Serial 10-fold dilutions of each
blood sample were prepared with phosphate-buffered saline
containing 1% albumin (pH 7); 0.1 ml of each dilution was
cultured on tryptic soy agar, and CFU were counted after
overnight incubation at 37°C.
Endotoxin assay. Free endotoxin was separated from bac-
terial cell-bound endotoxin by filtration as previously de-
scribed (13). Levels were quantitated with the Pyrotell
Limulus lysate gelation assay (Associates of Cape Cod,
Woods Hole, Mass.). Plasma inhibitors of the Limulus assay
were inactivated by diluting the plasma sample 1:3 with
pyrogen-free water and heating to 100°C for 10 min. Serial
twofold dilutions of plasma samples were assayed in parallel
with known concentrations of reference E. coli endotoxin
(Associates of Cape Cod). Results were expressed as the
amount of U.S. standard endotoxin with activity equivalent
to that of the plasma sample. The validity and specificity of
this assay for endotoxin has been previously confirmed (13).
Physiologic measurements. Mean arterial pressure, core
body temperature, and heart rate were continuously moni-
tored (Seimens 404), and recorded on a strip chart recorder
(Western Graphtec).
Statistical analysis. Student's t test was used to perform
statistical analysis of data with a model HP41-CV program-
mable calculator (Hewlett-Packard Co., Corvalis, Oreg.).
Significance was determined at P < 0.05.
RESULTS
Effect of methylprednisolone on bacteremia. Before the
administration of moxalactam, the geometric mean bacterial
counts were 8.5 x 103 and 3.1 x 104 CFU/ml of blood for the
methylprednisolone and placebo treatment groups, respec-
tively (Fig. 1A). The geometric mean levels of bacteremia
peaked 1 h after moxalactam administration (2.5 h after
infection) at 5.9 x 104 and 3.2 x 104 CFU/ml of blood for the
methylprednisolone and placebo treatment groups, respec-
tively. The geometric mean levels of bacteremia declined
throughout the remaining observation period in both groups.
Although the methylprednisolone-treated group exhibited a
trend toward higher levels of bacteremia, at no time were the
mean levels of bacteremia in the two groups significantly
different. Five animals, three in the placebo-treated group
and two in the methylprednisolone-treated group, com-
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

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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
TABLE 1. Effect of methylprednisolone administration on the
mean blood levels of leukocytes, platelets, and hemoglobin during
antibiotic therapy of experimental E. coli sepsis"
h after Results with placebo group/methylprednisolone group
bacterial 103 Hemoglobin
challenge Leukocytes/Il Platelets/t.l (g/100 ml)
0 4,700/3,800 303/329 11.6/11.4
1.5b 1,800c/2,300c 265/315 11.9/11.9
3.5 1,060c/730c 149/7c 12.2/11.0
5.5 1,000c/850c 38c/19c 9.4c/10.4
a None of the differences between treatment groups is statistically signifi-
cant.
b Methylprednisolone or
placebo was administered with moxalactam.
c Significantly different from the mean at time 0 as assessed by the
comparison of two means t test.
stood, proposed explanations include an intrinsic anti-
endotoxin activity of corticosteroids (1), the prevention of
endotoxin-induced hypoglycemia (5), improved circulation
and distribution of antibiotics (5), and the prevention of
complement-induced granulocyte aggregation (3). Enhanced
clearance of bacteremia or endotoxemia could also be a
beneficial factor, but these potential effects have been the
subject of few investigations. On the other hand, as
corticosteroids are known to decrease neutrophil phagocytic
function (4) and contribute to superinfection (15), corticoste-
roid administration could potentially result in decreased
clearance of bacteremia. Consistent with this idea Skornik
and Dressler reported that administration of corticosteroids
to both normal and burned rats acutely depressed clearance
of Pseudomonas aeruginosa from the lung (14). In the
present model of gram-negative bacterial sepsis, levels of
bacteremia were not significantly different between the
methylprednisolone and placebo treatment groups. Rather,
levels of bacteremia appeared to be influenced solely by
antibiotic administration (Fig. 1A).
In a recent report, Johnston and Greisman (8) examined
the effect of corticosteroid administration on endotoxin
levels in a murine model of gram-negative bacterial sepsis.
Mice that were treated with kanamycin alone had similar
plasma endotoxin levels but a higher mortality rate when
compared with mice that received kanamycin and
corticosteroids. In agreement with these findings, the pre-
sent data show no effect of methylprednisolone administra-
tion on the plasma levels of either total or free endotoxin.
However, a recent study indicates that dexamethasone
administration can alter uptake of free lipopolysaccharide by
the liver, spleen, adrenal gland, and lung without signifi-
cantly changing plasma levels of lipopolysaccharide (9).
Thus, clinically significant effects of corticosteroids medi-
ated by modulation of uptake of lipopolysaccharide by these
organs could potentially occur without significantly altered
plasma levels of lipopolysaccharide.
A criticism of some earlier studies with experimental
animal models is that the administration of very large doses
of gram-negative bacteria was also accompanied by a lethal
bolus of preformed endotoxin (1). Thus, rather than a model
of infection, these experiments merely assessed the ability of
corticosteroids to mitigate the effects of endotoxin. In the
present study, initial levels of plasma endotoxin were low
despite the intraperitoneal injection of large numbers of
bacteria. In fact, injection of equal numbers of bacteria
without mucin fails to induce either bacteremia or
endotoxemia (data not shown), indicating that intraperitone-
al injection of endotoxin has little or no effect in the present
29
model. However, after infection, bacteria present in blood
are lysed by the action of antibiotics, liberating large
amounts of endotoxin (13). This relation between bacterial
lysis and endotoxin liberation closely parallels observations
of bacterial clearance and endotoxin liberation in patients
receiving antibiotics for gram-negative bacterial sepsis (J. L.
Shenep, F. F. Barrett, G. L. Stidham, D. F. Westen-
kirchner, and P. Flynn, Crit. Care Med. 13:298, 1985).
Although corticosteroids clearly mitigate some of the toxic
effects of endotoxin, their impact in the therapy of gram-
negative bacterial sepsis is less clear. Despite the fact that
bacterial clearance and endotoxin liberation are readily
influenced in this model (12), the results of the current study
did not demonstrate an adverse effect of methylprednisolone
administration on bacterial clearance during antibiotic ther-
apy for gram-negative bacterial sepsis. On the other hand,
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this study revealed no beneficial effect of methylpredniso-
lone administration on either the level of endotoxin or on
endotoxin-sensitive physiologic, metabolic, or hematologic
parameters in the present model. To date, clinical studies of
the effectiveness of corticosteroid administration in prevent-
ing mortality in gram-negative bacterial sepsis are collec-
tively inconclusive. However, in one distinct subset of
patients, those with typhoid fever, a clear benefit appears to
result from corticosteroid administration (7). Further
progress in understanding both the pharmacologic and
microbiologic effects of corticosteroid administration during
therapy of gram-negative bacterial sepsis may eventually
permit delineation of other subsets of patients with gram-
negative bacterial infections that may benefit from cortico-
steroid administration.
ACKNOWLEDGMENTS
This work was supported by Public Health Service grant
RR-00584-20 from the Division of Research Resources, National
Institutes of Health, by grant CF-6040 from the American Cancer
Society, and by the American-Lebanese-Syrian Associated Chari-
ties.
LITERATURE CITED
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