Full text of DOI:10.1111/j.1348-0421.2002.tb02684.x

Microbiol. Immunol., 46(3), 181―186, 2002
Anti-CD11b Monoclonal Antibody Suppresses Brain
Dissemination of Cryptococcus neoformans
in Mice
,
1
1
1, ꢀ1
Kazuyoshi Kawakami* , Yoshinobu Koguchi , Mahboob Hossain Qureshi
,
1, ꢀ2
1
1
1
2
Tiantuo Zhang , Yuki Kinjo , Satomi Yara , Kaori Uezu , Kazutoshi Shibuya ,
Shiro Naoe , and Atsushi Saito
2
1
1
First Department of Internal Medicine, Faculty of Medicine, University of the Ryukyus, Okinawa 903
―
0215, Japan, and
2
Department of Pathology, Ohashi Hospital, Toho University School of Medicine, Ohta-ku, Tokyo 143
―8541, Japan
Received August 27, 2001; in revised form, November 30, 2001. Accepted December 10, 2001
Abstract: To elucidate the role of the ꢁ2 integrin family of adhesion molecules in the disseminated infection
of Cryptococcus neoformans from the lung to the central nervous system, we examined the effects of
monoclonal antibodies (mAbs) against CD11a, CD11b, CD11c and CD18 on the number of live microor-
ganisms in both the lung and brain of mice three weeks after intratracheal infection. Administration of anti-
CD11b mAb partially, but reproducibly, reduced the fungal loads in the brain in three independent
experiments, while the lung loads were not affected. In addition, the same treatment significantly decreased
the number of live microorganisms in the blood. In sharp contrast, the brain loads one week after intra-
venous injection with C. neoformans were not affected by treatment with anti-CD11b mAb. Finally,
administration of mAb against other adhesion molecules (CD11a, CD11c or CD18) failed to affect the fun-
gal loads in the brain as well as in the lung three weeks after intratracheal instillation, except for anti-CD18
mAb which rather increased the brain loads. Our results suggested that CD11b might be involved at least
in part in the process of fungal dissemination from lung to brain, although the significance of other ꢁ2 inte-
grin family adhesion molecules remains to be substantiated.
Key words: Cryptococcus neoformans, Disseminated infection, Brain, CD11b
Cryptococcus neoformans, an opportunistic fungal
pathogen, causes a life-threatening meningoencephalitis
in severely immunocompromised patients such as those
with acquired immunodeficiency syndrome (AIDS).
The host defense against this pathogen is mediated by
cellular immunity (19), and cytokines secreted from
type-1 helper T (Th1) cells play a central role in the
presence of defective IL-12 synthesis (13). However, the
precise mechanism that allows this fungal pathogen to
spread the infection from the lung to the brain remains to
be elucidated.
A variety of cell surface molecules are adopted by
pathogenic microorganisms for their entry into host
cells. Mac-1 (CD11b/CD18), a member of β2 integrin
adhesion molecules, is identified as a primary receptor for
several fungal pathogens including Candida albicans
(6), Histoplasma capsulatum (1), Blastomyces dermati-
tidis (21) and also C. neoformans (2, 17, 18). Dong
and Murphy (4) showed the direct binding of crypto-
coccal capsular polysaccharides to CD18 on human
neutrophils. In the present study, to determine the mech-
anism of dissemination of C. neoformans from the pri-
mary infected organ to the CNS, we investigated, using
a murine model of pulmonary cryptococcosis, the
establishment of protective immunity (3, 8
―
11, 13).
These mechanisms often succeed in localizing the infec-
tion within the lung by preventing the pathogenic organ-
ism from disseminating to the central nervous system
(CNS) and disseminated infection is accelerated in the
*
Address correspondence to Dr. Kazuyoshi Kawakami, The
First Department of Internal Medicine, Faculty of Medicine, Uni-
versity of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903
215, Japan. Fax:＋81 98 895 1414. E-mail: kawakami@med.
u-ryukyu.ac.jp
―
0
―
―
―
ꢀ1
Present addresses: Division of Infectious Diseases, Depart-
ment of Internal Medicine, University of Kentucky, Lexington,
Abbreviations: CNS, central nervous system; CR, comple-
ment receptor; mAb, monoclonal antibody; PDA, potato dextrose
agar; Th1 cells, type-1 helper T cells.
ꢀ
2
Kentucky 40536, U.S.A. Department of Internal Medicine,
Sun Yat-sen University of Medical Science, Guangzhou, China.
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K. KAWAKAMI ET AL
involvement of β2 integrins such as LFA-1, Mac-1 and
complement receptor type 4 (CR4) in this process by
examining the effects of the administration of specific
monoclonal antibodies (mAbs) against α and β chains of
these molecules.
tively, all purchased from ATCC]. Mice were injected
intraperitoneally with 200 µg each of mAb at days
―
3, 0,
＋3, ＋7 and ＋14 after infection with C. neoformans.
Rat IgG (ICN Pharmaceuticals, Inc., Aurora, Oh.,
U.S.A.) or hamster IgG (Organon Teknika Co., Durham,
N.C., U.S.A.) was used as the control Ab.
Materials and Methods
Preparation of pulmonary leukocytes. Pulmonary
intraparenchymal leukocytes were harvested as described
previously by our laboratory (7). The cells spun-down
onto the glass slides were stained with periodic acid-
Schiff (PAS) using a standard staining procedure, and
examined under a light microscope.
Immunohistochemical examination. Frozen sections
of lung were prepared from mice sacrificed 14 days
after intratracheal infection with C. neoformans. A
standard peroxidase/antiperoxidase technique was per-
formed on these sections using anti-CD11b mAb, and the
antigen-antibody complexes were visualized with
diaminobenzidine. Histopathological examination was
carried out to determine positive cells on the sections.
Statistical analysis. Differences between groups were
examined for statistical significance using ANOVA test
with a post-hoc analysis (Fisher PLSD test). A P value
less than 0.05 was considered significant.
Animals. Female (BALB/c×DBA/2) F1 mice were
purchased from SLC Japan (Hamamatsu, Japan) and
used in the present experiments at the age of 7 to 10
weeks. All mice were housed in a pathogen-free envi-
ronment and received sterilized food and water in the
Laboratory Animal Center for Biological Science, Uni-
versity of the Ryukyus. All experimental protocols
described in this study were approved by the Ethics
Review Committee for Animal Experimentation of our
university.
Cryptococcus neoformans. A serotype A-encapsulated
strain of C. neoformans, designated as YC-11, was
established from a patient with pulmonary cryptococ-
cosis. The yeast cells were cultured on potato dextrose
agar (PDA) plates for 2 to 3 days before use. This strain
rapidly multiplies in the lung and hematogenously dis-
seminates to the brain two or three weeks after intratra-
cheal instillation, and resulted in the death of infected
mice within six weeks (9).
Results and Discussion
Infection of microorganisms. Mice were anesthetized
by intraperitoneal injection of 70 mg/kg of pentobarbital
In our murine model of pulmonary cryptococcosis, the
fungal microbe disseminated from the lungs to the brain
and caused meningoencephalitis two to three weeks
after primary infection (9). To elucidate the mecha-
nism of such dissemination, we examined the effects
of mAbs against β2 integrin family adhesion molecules
such as CD11a, CD11b, CD11c and their common β
chain, CD18, on the brain loads of C. neoformans three
weeks after intratracheal infection. As shown in Fig. 1A,
in three independent experiments, anti-CD11b mAb par-
tially, but consistently, reduced the number of live
microorganisms in the brains, compared to mice treated
with control rat IgG, although the extent of reduction was
not profound. The same treatment did not affect the
proportion of CD11b＋ leukocytes, including macro-
phages and neutrophils, obtained from lungs in a flow
cytometric analysis (data not shown). In contrast, admin-
istration of mAbs against other adhesion molecules did
not change the infectious loads of C. neoformans in the
brain and lung (Fig. 2). These results suggested that
CD11b might be involved at least in part in the process of
fungal dissemination to the brain, although the signifi-
cance of other molecules remains to be substantiated.
It is possible that the attenuated brain dissemination by
anti-CD11b mAb was secondary to a reduction in the
fungal load in the lungs. However, this was not the
(Abbott Lab., North Chicago, Ill., U.S.A.) and restrained
on a small board. In each mouse, a 50-µl suspension of
5
normal saline containing live C. neoformans (1×10
yeast cells) was inoculated by inserting a 25-gauge blunt
needle into and parallel to the trachea. In some experi-
ments, the same number of microorganisms was intra-
venously inoculated at 100 µl per mouse under ether
anesthesia.
Enumeration of viable C. neoformans. Mice were sac-
rificed three weeks after intratracheal infection or one
week after intravenous infection, and lungs and brains
were homogenized separately in 10 ml of distilled water
by teasing with a stainless mesh. The homogenates,
appropriately diluted with distilled water, were inoculated
at 100 µl on PDA plates, cultured for 2 to 3 days fol-
lowed by counting the number of colonies. In some
experiments, the fungal loads in heparinized peripheral
blood were also examined.
Antibodies. Anti-CD11a, -CD11b, -CD11c and -CD18
mAbs were purified by a protein G column kit
(
Kirkegaard & Perry Lab., Gaithersburg, Md., U.S.A.)
from serum-free culture supernatants of hybridomas
clones M17/5.2 (rat IgG), M1/70.15.11.5.HL (rat IgG),
N418 (hamster IgG) and 2E6 (hamster IgG), respec-
[

ANTI-CD11b mAb INHIBITS BRAIN DISSEMINATION OF C. NEOFORMANS
183
(A)
(B)
Fig. 1. Anti-CD11b mAb reduces C. neoformans counts in the brain, but not in the lung, of intratracheally infected mice. Mice
received intraperitoneal injections of anti-CD11b mAb or control rat IgG (200 µg/mouse) at days －3, 0, 3, 7 and 14 after intratracheal
5
infection with C. neoformans (1×10 /mouse). Three weeks later, mice were sacrificed and the number of live microorganisms in brain
(A) and lung (B) were counted. Each symbol represents the result of a single mouse and the symbols within shaded area represent mice
with zero counts. *, P＜0.05; NS, not significant.
case, because administration of this mAb did not exert
any significant effect on the number of live microor-
ganisms in the lung (Fig. 1B). To further elucidate the
mechanism of inhibition of brain dissemination, we
examined the effect of anti-CD11b mAb on the fungal
loads in the blood of mice infected intratracheally with C.
neoformans. In addition, the effect of this treatment on
the brain loads in mice infected through intravenous
injection of the microorganism was examined. As shown
in Fig. 3, administration of anti-CD11b mAb signifi-
cantly reduced the number of live fungal microorganisms
in the blood compared to that in mice treated with control
rat IgG three weeks after intratracheal infection. In
sharp contrast, infectious loads of C. neoformans in the
brain were not significantly affected by administration of
anti-CD11b mAb compared to mice treated with control

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K. KAWAKAMI ET AL
Fig. 2. Anti-CD11a, -CD11c and -CD18 mAb fail to reduce infectious loads of C. neoformans in the brain of intratracheally infected mice.
Mice received intraperitoneal injections of anti-CD11a, -CD11c, -CD18 mAb or control rat or hamster IgG (200 µg/mouse) at days 3,
, 3, 7 and 14 after intratracheal infection with C. neoformans (1×10 /mouse). Three weeks later, mice were sacrificed and the num-
―
5
0
ber of live microorganisms in brain was counted. Each symbol represents the result of a single mouse and the symbols within shaded
area represent mice with zero counts. The experiments were repeated three times with similar results. *, P＜0.05; NS, not significant.
Table 1. Anti-CD11b mAb fails to reduce infectious loads of C.
a)
neoformans in the brain of intravenously infected mice
b)
Treatments
C. neoformans (Log10 CFU/brain)
PBS
Rat IgG
Anti-CD11b mAb
6.87±0.09
c)
6.86±0.08
c)
6.77±0.19
a)
Mice received intraperitoneal injections of anti-CD11b mAb
or control rat IgG (200 µg/mouse) at days －3, 0 and 3 after intra-
5
venous injection with C. neoformans (1×10 /mouse). One week
later, mice were sacrificed and the number of live microorganisms
in brain was counted.
b)
Mean±SD of seven mice.
Not significantly different from PBS-treated mice.
c)
the translocation of C. neoformans from the lung to
blood, but not from blood to brain.
Fig. 3. Anti-CD11b mAb reduces infectious loads of C. neofor-
mans in the circulation of intratracheally infected mice. Mice
received intraperitoneal injections of anti-CD11b mAb or control
rat IgG (200 µg/mouse) at days －3, 0, 3, 7 and 14 after intra-
In preliminary experiments, we determined the num-
ber of live C. neoformans in the lung, blood and brain of
mice instilled intratracheally with the microorganism
at various doses in order to elucidate the relationship in
fungal loads among these organs. The results indicated
that the number of live microorganisms in the lung did
not correlate with that in the brain nor in the blood,
three weeks after infection [r＝0.24, P＞0.1 (n＝23)
and r＝0.21, P＞0.1 (n＝17), respectively] (data not
shown). In some mice, a large number of fungal
microorganisms were detected in the brain although
only a few were found in the lungs. In other mice bear-
ing large numbers of microorganisms in their lungs,
5
tracheal infection with C. neoformans (1×10 /mouse). Three
weeks later, mice were sacrificed and the number of live microor-
ganisms in blood was counted. Each symbol represents the result
of a single mouse and the symbols within shaded area represent
mice with zero counts. The experiments were repeated three
times with similar results. *P＜0.05.
rat IgG at day 7 after intravenous infection (Table 1).
Considered collectively, these results clearly demon-
strate that the α chain of Mac-1 molecule is involved in

ANTI-CD11b mAb INHIBITS BRAIN DISSEMINATION OF C. NEOFORMANS
185
very few or none disseminated to the brain. In sharp con-
trast, there was a significant relationship in fungal loads
between blood and brain [r＝0.75, P＜0.001 (n＝17)].
These data suggest that the translocation of C. neofor-
mans from the lungs to bloodstream cannot simply be
explained by physical mechanisms, i.e., mechanical
pressure on alveolar walls by overgrowing yeast cells.
Rather, such translocation is probably controlled by
active processes such as receptor-mediated binding.
This conclusion is in agreement with the present findings
indicating the involvement of Mac-1-mediated mecha-
nism in fungal translocation from the lung to blood, but
not from blood to brain.
C. neoformans activates the alternative complement
pathway (14, 16), which results in the deposition of C3
fragments on the surface of the microorganism, mostly in
the form of iC3b (15, 16). iC3b acts as the major opson-
ic component for phagocytosis of this microorganism by
phagocytic leukocytes through binding to CR3 (Mac-1)
first week and 1.2±0.1% vs. 0.9±0.4% (n＝3 each)
after the second week]. Thus, in the present study, the
suppressive effect of this mAb on the brain dissemination
of C. neoformans was not ascribed to the hypothetical
mechanism described above.
In an alternative explanation, anti-CD11b mAb treat-
ment might cause some positive change in the cytokine
production by macrophages, leading to the enhanced
host defense against C. neoformans in the lung and
reduced dissemination to the brain. However, this was
not likely to be the case because the clearance of microor-
ganisms from the lungs was not accelerated by this
treatment. The local host defense in the lung might
rather be attenuated, because IL-12 production by
macrophages is strongly inhibited in vitro by treatment
with anti-CD11b mAb (20, our unpublished results),
which should hamper the development of Th1 response.
Furthermore, in our mouse model of cryptococcal infec-
tion, Th1 response was strongly reduced before anti-
CD11b mAb treatment (10), which may result from the
attenuation of macrophage IL-12 synthesis by C. neo-
formans (12). Thus, further studies will be necessary to
understand the precise mechanism of the preventive
effect of anti-CD11b mAb on the disseminated infection
to the brain.
(2, 17, 18). In the present study, the mAb that recognizes
the α chain (CD11b) of CR3 significantly suppressed the
dissemination of C. neoformans from the lung to the
brain. Anti-CD11b mAb with the same clone name
(M1/70) was reported to decrease phagocytosis of Lis-
teria monocytogenes by macrophages to a level observed
in the absence of active complement (5). These findings
suggest that the opsonic binding of C. neoformans to
phagocytes and their subsequent ingestion by these cells
may be involved in the translocation of these microor-
ganisms from the alveolar space to the peripheral circu-
lation, that is, the phagocytes may function as carriers of
such transmigration. Compatibly with the above hypoth-
esis, C. neoformans strain used in the present study was
active in complement activation, as indicated by the
production of C3a fragment in human plasma, and alve-
olar macrophage was the only cell population positively
stained by this mAb in our immunohistochemical analy-
sis (data not shown).
In conclusion, we demonstrated in the present study
the possible involvement of CD11b in the disseminated
infection of C. neoformans from the lung to the brain.
Our data should be useful for the development of new
strategies aimed at limiting the infection of pathogenic
fungus within the primary infected organ.
This work was supported in part by Grants-in-Aid for Science
Research (C) (09670292 and 12670261) from the Ministry of
Education, Science, Sports and Culture, Japan and by a grant (H-
1
2-AIDS-004) from the Ministry of Health and Welfare, Japan.
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