Suppression of Cell-Mediated Immunity by Metronidazole

lnternational Archives of Allergy and Applied Immunology
Editors-in-Chief: R. R. A. Coor,os, Cambridge; P. Keu6s, Helsingborg; L. M. LTcHTENsrBN, Baltimore, Md.; F. Mn-cnou, Buffalo, N. Y.; Z. TnNre, Basel; G. B. Wesr, Epsom
Publishers: S. Kencrn, Basel
REPRDTT (Printed in Switzerland)
Int. Archs Allergy appl. Immun. 54: 422-427 (lW7)
Suppression of Cell-Mediated Immunity by Metronidazole
D.I.
Grove, A. A. F. Mahmoud and K. S.Warren
Division of Geographic Medicine, Department of Medicine, Case Western Reserve University and
University Hospitals, Cleveland, Ohio
Abstract. Metronidazole administered orally
in
doses
of 20 and 200 mg/kg
daily
suppressed granuloma formation around Schistosoma mansoni eggs which were injected
intravenously and lodged in the pulmonary rnicrovasculature of mice. The same doses did
not suppress granuloma formation in animals which had previously been sensitized to the
eggs. Nonspecific granulomatous inflammation around divinyl benzene copolymer beads
was unaJfected by the drug. In a daily dose of 20 mglkg, metronidazole inhibited delayed
footpad reactions to soluble schistosonne egg antigen, but 200 mgikg on alternate days
failed to suppress skin allograft rejection. The drug appears to suppress selectively some
aspects of cell-mediated immunity.
Both metronidazole and niridazole have
been reported to be effective for the treatment of guinea worm (Dracunculus medinesrs) infection, apparently acting by suppression of inflammation thus facilitating the removal of long segments of the worm [1, 3,
B]. Recently, it was shown that niridazole
suppresses cell-mediated immunological reactions including granuloma formation
around eggs of Schistosoma mansoni, deIayed footpad swelling following the injection of soluble schistosome egg antigens and
skin allograft rejection [5, 6]. These observations suggested the possibility that metronidazole may also have imrnunosuppressive
properties. The present studies were undertaken, therefore, to assess the effect of this
drug on the following cell-mediated immu-
nological reactions: granuloma formation
around S. mansoni eggs, nonspecific inflammation around divinyl benzene copolymer
beads, delayed hypersensitivity footpad reactions and skin allograft rejection.
Materials and Methods
Metronidazole was obtained from Searle (San
Juan, Puerto Rico). Different concentration of the
drug were suspended in distilled water and 0.4 ml
was administered to mice orally through a blunt
tip 16-gauge needle. Metronidazole is usually well
absorbed after oral administration [9].
Granuloma Formation around S. mansoni Eggs
Schistosome eggs were obtained from the livers
of mice 8 weeks after exposure to 200 cercariae of
a Puerto Rican strain of S. mansoni using the
423
Grove/MahmoudflVarren
method described by Moore et al. U\. Young fernale Swiss albino mice (Flow Laboratories, Dubiin, Virginia), 20-22 e in weight, were given 1,500
eggs intravenously via a tail vein. Two groups of
mice were used: unsensitized and sensitized, sensitization being achieved by intraperitoneal injection
of 1,500 eggs 14 days prior to the intravenous
injection of eggs. Administration of metronidazole
was begun on the day of intravenous egg injection. Eight days after intravenous egg injection in
the case of sensitized mice, and 16 days for unsensitized animals, groups of mice were killed, the
iungs removed, sectioned and stained as previously described [6]. Lungs were removed at these
times as they have been shown to be the points of
maximal inflammation around eggs in sensitized
and unsensitized animals, respectively [15]. Mea-
surement of the area of granulomatous inflammaeggs was performed with a zr-MC par-
difference in thickness at 24 h between the two experimental footpads of each animal was taken as
the net swelling.
Skin Allogralting
Adult female BALB/c J and C57BL/6 J mice
were obtained from Jackson Laboratories (Bar
Harbor, Me.). All mice were maintained on a diet
of commercial food pellets and water ad libitum'
Grafting of C57BL|6 I skin on to BALB/c J mice
was performed and graft survival scored as previously described [6]' Groups of mice were treated
with either 20 mg/kg metronidazole daily or
200 mg/kg on alternate days beginning 1 day before grafting.
Results
tion around
ticle
measurement computer system (Millipore
Corporation, Bedford, Mass.) as previously described 16l. Ten lesions from each of B or 9 animals were measured, the results tested statistically
by analysis of variance [11] and presented in the
tables as the mean value and range representing
950/o of the observed values.
G
rantdoma F ormation around Divinyl
Divinyl benzene copolymer plastic beads (BioRad, Richmond, Calif.) were prepared to a size
equivalent to schistosome eggs by screening
through a 70-mesh steel sieve and were
three times with normal saline [4]. Groups of mice
washed
were given either 200 or 20 mg/kg daily for 3
days, beginning 1 day before the injection of
in
0.5
The effect
E
gg Gr anuloma
of daily administration of
varying doses of metronidazole on the area
of granulomatous inflammation in unsensitized mice 16 days after intravenous egg
injection is shown in table I and figure 1'
There was increasing suppression of inflammation with increasing doses of metronida-
Benz.ene CopolYmer Beads
3,000 beads suspended
Schistosome
ml physiological
sa-
line via the tail vein. Lungs were removed at 48 h'
the time of the peak inflammatory reaction [4]
and measured as described above.
Delayed Footpad Swelling
zole, the results being statistically significant
in the animals which received 200 (p<0.01)
or z\mglkg (p(0.05), but not in
those
which were given 2.0 or O.Zmglkg daily' A
single dose of metronidazole 20A mg/kg on
the day of egg injection did not suppress the
granulornatous reaction (table I).
Metronidazole 200 mg/kg daily had no
effect on the mean granuloma size 8 days
after intravenous egg injection in sensitized
mice, the mearl area of inflammation being
(13,000-58,000 ,amz) compared
Mice were sensitized by the intraperitoneal
injection of 2,000 S. mansoni eggs and half were
35,400
given 20 mg/kg metronidazole daily from that day'
control mice.
In order to determine whether this failure to suppress inflammation in sensitized
mice was due to the fact that metronidazole
was administered for only 8 days (cf' L6
The effect of the drug on footpad swelling was
tested on the 14th day by injection of soluble
schistosome egg antigen equal to 10 pg protein in
ml as described [2], in one rear footpad, and
phosphate-buffered saline in the other. The mean
0.03
pcmz
with 39,500 pm2 (l-9,000-60,000 pm') in
Grove/Mahmoud/Warren
424
intravenous inTable I. Effect of different doses of metronidazole on granuloma formation 16 days after the
given
daily for 16
was
metronidazole
mice;
of
jection of S. msnsoni eggs into the pulmonary microvasculature
days
Control
Metronidazole,
Metronidazole,
Metronidazole,
Metronidazole,
Metronidazole,
n
t
200 mg/kg dailY
20 mg/ks dailY
2 mg/kg daiiY
0.2 mg/kg dailY
200 mg/kg once
areaa
Number
Mean
prfiz
9
16,000
8
9
4,500
9,100
I
9,700
8
16,300
8
17,500
Range, Pm2
5,600-26,700
700-8,300
700-21,500
1,700-23,800
1,900-33,300
5,000-30,100
Probabilityb
<0.01
<0.05
NS
NS
NS
Egg included.
Analysis of variance'
mice were given either 20 or 200 mg/kg me-
-E
tronidazole daily for 16 days. 1,500 eggs
were injected intravenously on the 8th day
of drug administration and the lungs removed on the 16th day. Again, no significant differences were found, the mean area
of inflammation being 30,800 tr,lm2 (6,85062,000 ptmz) in mice given 20 mglkg
metronidazole and 30,000 prm2 (7,50060,000 pmz) in those given 200 mg/kg
sco
d
o
400
e
6
c
.9
0
soo
d
t]
?nn
.q
metronidazole,
E
Po
roo
L9
cd
_s in
0.2
2
20
2C0
Metronidazote) mg/kg daiiY
Fig. 1. Effect of increasing doses of metronidazole on the mean granuloma area expressed as a
percentage of the area of the egg alone 16 days
after intravenous injection of S. mansoni eggs into
the pulmonary microvasculature of mice.
unsensitized mice), a further experiment was performed. 30 mice were sen-
days
cf.
25,000 pmz (3,600-
57,000 pm2) in control mice.
for
sitized by the intraperitoneal injection of
1,500 eggs. Two tleeks later, groups of 10
Plastic Bead Granulomq
Daily doses of metronidazole had no ef-
fect on the inflammatory reaction around
divinyl benzene copolymer beads 48 h after
intravenous injection. The mean area of inflammation in eight control mice was
4,400 prm2 (range 800-11-,000). This value
was not significantly different from the values of 3,600 (800-8,800) and 3,700 prm2
(600-7,600) for groups of eight mice given,
respectively, 200 and 20 mglkg metronidazole daily.
In
order to determine whether this fail-
Immunosuppression with Metronidazole
425
ure to suppress inflammation around beads
mediated immunity. Its influence was most
was due to the fact that metronidazole was
administered for only 48 h (cf. 16 days for
the egg granuloma in unsensitized mice), a
further experiment was performed. Groups
of ten mice were given either 20 or 200 mgl
kg metronidazole daily for L6 days. On the
74th day, 3,000 beads were injected intravenously into these mice and into ten con-
marked when given contemporaneously
with exposure to antigen and administered
trol mice. Again, no significant differences
were found, the mean area of inflammation
being 2,100 pm, (490-4,560 pm2) in mice
given 20 mglkg metronidazole and
L,970 p.m2 (640-4,340 pm2) in those given
200 mg/kg metronidazole,
2,720 prmz
(600-4,390,r2m2) in control mice.
cf.
Delayed
F
ootpad Swelling
Metronidazole 20 mgikg given daily sup-
of delayed hyperin response to
soluble schistosome egg antigen: control
mice had a mean increase in footpad thickness of 360+23 p.m compared with
34+44 p.m in the treated group (p(0.001",
pressed the development
sensitivity footpad swellings
Student's t test).
S
kin Allo gralt
Surv iv
al
daily thereafter.
A
dose-response effect was
demonstrated with the area of granulomatous inflammation around schistosome eggs
decreasing with increasing doses of metronidazole. Under these circumstances, the suppression of granuloma formation around
schistosome eggs was nearly as marked as
In a preliminary experiment, mice given 20 mg/kg of metronidazole daily had 47010 suppression of granuloma formation compared with 690/o suppression in those given 100 mg/kg of niridazole daily. In contrast to niridazole [5],
however, when metronidazole was given to
mice which were previously sensitized to antigen, no significant suppression of the granulomatous reaction occurred. Similarly, the
immunosuppressive action of metronidazole
was not long-lived as there was no eftect L6
days after a single administration whereas a
single dose of niridazole suppressed granuloma formation for more than 30 days [6].
That granuloma suppression is not a nonspecific, anti-inflammatory effect is indicated by the failure of metronidazole to affect
the nonimmunological, chemically-mediated
with niridazole.
BALB/c J mice treated with metronidazole did not show any prolongation of allograft survival. Grafts survived 10.4+0.2
days in 13 control mice compared with
10.3+0.2 days in nine mice given 20 mg/kg
daily and 10.6+0.2 days in 15 mice given
The inhibition of delayed footpad reactions to soluble schistosome egg antigen is
further evidence of the ability of metronidazole to suppress cell-mediated immunologi-
200 mglkg metronidazole on alternate days.
cal reactions. The inability of the drug to
Discussion
Metronidazole appears to have selective
immunosuppressive activity with inhibition
of some, but not other, parameters of cell-
plastic bead granuloma [4, 15].
suppress skin allograft rejection, even when
given prior to grafting, suggests that the immunosuppressive action of this drug is
selective. It must be noted, however, that
6-mercaptopurine, which is used widely in
clinical organ transplantation, failed to pro-
long skin allograft survival in mice
1l2l.It
Grove/Mahmoud/Warren
426
is possible that these various reactions may
be mediated by different subclasses of T
lymphocytes perhaps with varying cooperation of macrophages.
The immunosuppressive effects of metronidazole may be related to other phenomena which have been associated with
this drug. The increased incidence of lung
tumors and malignant lymphoma in mice
given metronidazole may be the result of inhibition of tumor surveillance systems [1-0].
Similarly, the rapid resolution of inflammation seen in patieqts with dracunculiasis
may, in part, reflect the irnmunosuppressive
activity of metronidazole f1, 3]. Furthermore, metronidazole reportedly hastened
the resolution of a variety of cutaneous ulcers, and it was suggested that this was due
to an anti-inflammatory action [13].
A recent report has indicated that metronidazole may be beneficial in patients
with Crohn's disease [1a]. In the absence of
any known immunosuppressive action,
these authors suggested that the drug may
exert its effect by altering the bacterial flora
of the gut. In view of the fact that Crohn's
disease is characterized pathologically by
granuloma formation, this beneficial response may be due in part, to an immunosuppressant effect of metronidazole. Further
investigations of the anti-inflammatory and
immunosuppressive properties of this drug
may be fruitful.
References
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Acknowledgements
We gratefully acknowledge the expert technical assistance of Melanie Zuik, Earlene Moss and
Pierre A. Peters. "fhese studies have been supported by a grant from NIH-PHS No. AI-08163 and
grants from The Rockefeller and Edna McConnell
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Correspondence to: Dr. David I. Grove,
Division of Geographic Medicine,
Wearn Research Building, University Hospitals,
Cleveland, OH 44106 (USA)