Tamm-Horsfall glycoprotein (THP) contains manno-oligosaccharides that are recognized by type 1 fimbriae (F1) of Escherichia coli. In the present study, we examined the in vivo phagocytic activity of mouse peritoneal macrophages after treatment of bacteria with THP. At low THP concentrations (12.5 microg/ml and 50 microg/ml) no significant difference was observed in the phagocytosis of E. coli F1+. However, at high THP concentrations (500 microg/ml and 1250 microg/ml) we obtained a reduction of bacterial phagocytosis by mouse peritoneal macrophages.34913-

Bastos, A C

Oliveira, U M

Santos, L B

Tamashiro, W M

Yamada, A T

Yano, T

Brazilian Journal of Medical and Biological Research

English

Repositorio da Producao Cientifica e Intelectual da Unicamp

913
Braz J Med Biol Res 34(7) 2001
Role of THP on phagocytosis of E. coliBrazilian Journal of Medical and Biological Research (2001) 34: 913-917
ISSN 0100-879X
Role of Tamm-Horsfall protein in the
binding and in vivo phagocytosis of
type 1 fimbriated Escherichia coli by
mouse peritoneal macrophages
Departamentos de 1Microbiologia e Imunologia and
2Histologia e Embriologia, Instituto de Biologia, and
3Patologia Clínica, Faculdade de Medicina,
Universidade Estadual de Campinas, Campinas, SP, Brasil
A.C.S.C. Bastos1, L.B. Santos1,
W.M.S.C. Tamashiro1,
A.T. Yamada2, U.M. Oliveira3
and T. Yano1
Abstract
Tamm-Horsfall glycoprotein (THP) contains manno-oligosaccharides
that are recognized by type 1 fimbriae (F1) of Escherichia coli. In the
present study, we examined the in vivo phagocytic activity of mouse
peritoneal macrophages after treatment of bacteria with THP. At low
THP concentrations (12.5 µg/ml and 50 µg/ml) no significant differ-
ence was observed in the phagocytosis of E. coli F1+. However, at high
THP concentrations (500 µg/ml and 1250 µg/ml) we obtained a
reduction of bacterial phagocytosis by mouse peritoneal macrophages.
Correspondence
T. Yano
Departamento de Microbiologia e
Imunologia, IB, Unicamp
13083-970 Campinas, SP
Brasil
Fax: +55-19-3788-8190
E-mail: tyano@obelix.com.br
Research supported by FAPESP.
Received May 30, 2000
Accepted April 10, 2001
Key words
· Tamm-Horsfall protein
· Type 1 fimbriated E. coli
· Phagocytosis
Introduction
Several members of the Enterobacteria-
ceae family, including some strains of Esch-
erichia coli, have numerous filamentous fim-
brial structures on their surface (1). Some
strains of type 1 fimbriated E. coli adhere to
uroepithelial cells and may cause lower uri-
nary tract infections (cystitis) (2), but their
relationship with upper urinary tract infec-
tions (pyelonephritis) is still not clear. These
fimbriae facilitate the attachment of bacteria
to mannose-containing receptors on a vari-
ety of host cells, as well as to a urinary tract
glycoprotein known as Tamm-Horsfall pro-
tein (THP) (3-5).
Tamm-Horsfall glycoprotein, which is
synthesized exclusively by the kidney (6), is
present in large quantities (20-200 mg/ml) in
human urine and has been implicated in a
variety of renal diseases, although its physi-
ological role is unknown (7). The attachment
of type 1 fimbriated E. coli to THP suggests
a protective role of THP in the defense against
urinary tract colonization and infection by
this strain of E. coli (3,5). On the other hand,
an in vitro study (8) demonstrated that bacte-
ria coated with THP are less susceptible to
phagocytosis by polymorphonuclear leuko-
cytes (PMNL), and this may allow the infec-
tion to persist. Type 1 fimbriae can also bind
to mannose-containing receptors on the mem-
branes of PMNL and mouse peritoneal mac-
rophages (9).
The aim of the present study was to ex-
amine in an in vivo assay the effect of THP
concentration on bacterial phagocytosis by
mouse peritoneal cavity macrophages, a phe-
nomenon that has only been described in
vitro by others.
914
Braz J Med Biol Res 34(7) 2001
A.C.S.C. Bastos et al.
Material and Methods
Bacterial strains
E. coli strain ORN115, a recombinant
with type 1 fimbriae (10), was obtained from
Dr. Paul E. Orndorff (Department of Micro-
biology, Pathology, and Parasitology, North
Carolina State University, Raleigh, NC,
USA). Strains of non-fimbriated E. coli (K12
C600) were also used. The bacteria were
cultured statically in trypticase soy broth for
18 h at 37oC (11). The presence of type 1
fimbriae was confirmed by serum agglutina-
tion on glass slides with specific antiserum
against F1+ and by agglutination of guinea
pig erythrocytes on slides.
Purification of THP
THP from human urine was purified by
salt precipitation (12). After dialysis against
deionized water and lyophilization, the ma-
terial was dissolved in 8 M urea and chro-
matographed on a Sepharose CL-6B column
(2 cm x 58.6 cm) equilibrated with 30 mM
phosphate buffer, pH 6.8, containing 2 M
urea at a flow rate of 2 ml/min. The elution
profile was monitored at 280 nm. The active
peak appeared as a single band (molecular
weight ~94,000) on SDS-PAGE (13) after
staining with silver nitrate (14). This band
was positively identified as THP by Western
blotting (15) following incubation with an
anti-THP monoclonal antibody (Accurate
Chemical and Scientific, Westbury, NY,
USA). Protein concentrations during purifi-
cation were determined using a Bio-Rad pro-
tein assay kit (Bio-Rad Laboratories, Her-
cules, CA, USA).
Preparation of anti-THP serum
Polyclonal anti-THP antiserum was pro-
duced in New Zealand rabbits as described
by Dawnay et al. (16), with some modifica-
tions. The rabbits were immunized subcuta-
neously at various sites with 250 µg of THP
in Freunds complete adjuvant. After 30 and
60 days, the animals received an additional
injection of 100 µg of THP in Freunds
incomplete adjuvant and were bled 10 days
after the final dose.
Binding of E. coli to immobilized THP
The methodology developed by Karlsson
et al. (17) was used. After SDS-PAGE (13),
THP was transferred to a nitrocellulose mem-
brane (15) and blocked by incubation with
5% non-fat dry milk in 20 mM PBS, pH 7.4,
at room temperature for 1 h. The membrane
was then washed twice in 20 mM PBS-
Tween buffer, pH 7.4, and incubated in a
vertical position in 10 ml of a bacterial sus-
pension (108 cells/ml) of E. coli ORN115
and E. coli K12 C600 for 1 h at room temper-
ature with no shaking. A control sample
containing 20 mg/ml D-mannose was also
run. After this period, the membranes were
washed in 20 mM PBS-Tween buffer and
placed protein side up on eosin blue methyl-
ene (EMB) agar plates and incubated at 37oC
for 18 h to allow colony formation.
Phagocytosis assay
Two hundred microliters of E. coli
ORN115 suspension (108 cells/ml in 20 mM
PBS, pH 7.4) was centrifuged at 17,700 g
and resuspended in 20 mM PBS, pH 7.4,
containing THP (2.5, 25, 100 and 250 µg/0.2
ml (8)) and incubated for 30 min at 37oC.
The peritoneal cavities of 40 female 8-
10-week-old BALB/c mice supplied by the
Universitys central animal house (CEMIB/
Unicamp) were stimulated by injecting a
10% peptone solution (18) (Difco Laborato-
ries, Detroit, MI, USA) (0.5 ml/animal). Three
days later, a control group of eight mice were
injected intraperitoneally (ip) with 0.2 ml of
E. coli (108 cells/ml in 20 mM PBS, pH 7.4
(19)) and four other groups received a 0.2-
ml suspension of E. coli (108 cells/ml) con-
915
Braz J Med Biol Res 34(7) 2001
Role of THP on phagocytosis of E. coli
taining 2.5, 25, 100 and 250 µg of THP/0.2
ml, respectively. Thirty minutes after the ip
injections (18), the mice were sacrificed by
cervical dislocation, and their peritoneal cavi-
ties were washed with 3 ml of 20 mM PBS
containing 5 IU heparin/ml. The macrophages
collected were resuspended to 2 x 106 cells/
ml in 20 mM PBS, pH 7.4. This suspension
was cytocentrifuged and the cells were
stained with Giemsa (8). The percentage of
phagocytes was determined by counting
phagocytic and non-phagocytic cells in a
total of 100 cells. The numbers of macro-
phages were counted by light microscopy at
a magnification of 100X. Differences be-
tween the experimental and control groups
were compared by the Duncan multiple range
test using a statistical software package (SAS
Institute, 1986) (20).
Results
Characterization of THP
Chromatography of THP on Sepharose
CL-6B in 8 M urea produced two main peaks,
the first of which contained THP (data not
shown). After applying SDS-PAGE and stain-
ing with silver nitrate (Figure 1), THP was
detected as a single band of 94 kDa. Identical
results were obtained by Western blotting
(Figure 2).
Binding of E. coli to immobilized THP
Type 1 fimbriated E. coli bound to immo-
bilized THP on nitrocellulose membranes (data
not shown). This binding was observed after
multiplication of E. coli around the 94-kDa
THP band on EMB agar plates. No growth was
observed in the presence of D-mannose (20
mg/ml) or with E. coli K12 C600.
Phagocytosis
Duncans multiple comparisons test re-
vealed significant differences (P<0.0001) in
Figure 1. SDS-PAGE of purified
Tamm-Horsfall protein (THP).
Proteins were loaded onto 10%
polyacrylamide gels in the pres-
ence of a-mercaptoethanol and
stained with silver nitrate. Lane
A, Molecular mass standards of
thyroglobulin (330 kDa), ferritin-
half unit (220 kDa), bovine albu-
min (67 kDa), catalase (60 kDa),
lactate dehydrogenase (36 kDa),
and ferritin (18.5 kDa); lanes B-
D, THP purified by gel filtration
chromatography on a Sepharose
CL-6B column; lane E, standard
THP; lane F, molecular mass
standards of phosphorylase B (94 kDa), bovine albumin (67 kDa), ovalbumin (43 kDa),
carbonic anhydrase (30 kDa), trypsin (20.1 kDa), and a-lactalbumin (14.4 kDa).
kDa
330
kDa
220
67
60
36
18.5
94
67
43
30
20.1
A B C D E
Figure 2. Western blotting of pu-
rified Tamm-Horsfall protein
(THP). Lane A, Molecular mass
standards (Pharmacia). Lanes B
and C, THP purified by gel filtra-
tion and detected with mouse
monoclonal anti-THP (Accurate
Chemical and Scientific) and rab-
bit antiserum, respectively. Lane
D, Standard THP detected with
mouse monoclonal anti-THP,
and lane E, standard THP de-
tected with rabbit polyclonal
anti-THP.
kDa
94
67
43
30
20.1
A B C ED
14.4
the phagocytosis of bacteria associated with
THP. When the bacteria were preincubated
with THP (2.5 µg or 10 µg/0.2 ml) there were
no significant differences in phagocytosis
compared to bacterial suspensions without
Table 1. Percentage of E. coli F1+ phagocytosis by
mouse peritoneal macrophages after incubation
of bacteria with Tamm-Horsfall protein (THP).
THP (µg/0.2 ml) Phagocytosis (%)
0 97.8
2.5 97.0
10 96.7
100 86.8
250 82.2
Differences between the experimental and con-
trol groups were compared by the Duncan mul-
tiple range test using a statistical software pack-
age (Ref. 20) (P = 0.0001; F = 33.82).
14.4
F
916
Braz J Med Biol Res 34(7) 2001
A.C.S.C. Bastos et al.
THP (Table 1). However, when the concen-
tration of THP was increased to 100 µg and
250 µg/0.2 ml phagocytosis was significant-
ly decreased.
Discussion
The ability of bacteria to bind to epitheli-
al surfaces through fimbriae is correlated
with their pathogenicity (1). Some strains of
E. coli isolated from patients with lower
urinary tract infections express type 1 fim-
briae (2). These structures allow the bacteria
to adhere to receptors containing mannose
on the surface of host epithelial cells (3) and
phagocytes (19). Glycoproteins present in
urine can function as receptors for type 1
fimbriae and influence bacterial adhesion to
the uroepithelium and to phagocytes (6,8).
As shown here, the urinary glycoprotein
THP, which contains the sugar D-mannose,
functions as a mannose-sensitive receptor
for type 1 fimbriated E. coli. These results
agree with the studies of Orskov (3) who
suggested this binding to be mannose-sensi-
tive. We observed this interaction at low
THP concentrations (5 µg/ml), although oth-
ers (21) have reported this effect only at THP
concentrations up to 100 µg/ml. These same
investigators observed that the receptor for
F1+ in urine was a low molecular weight
substance. This contrasts with our results
and previous reports (22) which character-
ized THP as a high molecular weight (94,000)
glycoprotein.
THP may act as a competitive inhibitor of
the binding of E. coli F1+ to PMNL (8). PMNL
are important cells in the initial stages of
infection, before the formation of specific
antibodies when serum opsonin levels are low.
We used mouse peritoneal macrophages
to assess the effects of THP on the phagocy-
tosis of E. coli F1+. The phagocytosis of E.
coli F1+ varied with THP concentration.
When E. coli F1+ was preincubated with
high THP concentrations (100 µg or 250 µg/
0.2 ml) the phagocytosis of bacteria by mac-
rophages decreased. In contrast, when E.
coli F1+ was preincubated with lower THP
concentrations (2.5 µg and 10 µg/0.2 ml)
there was no significant effect on phagocy-
tosis.
Horton et al. (23) suggested that THP
may stimulate phagocytosis since in aggre-
gated form and at high concentrations (>500
µg THP/ml) it increased neutrophil phago-
cytosis and lysosome degranulation. How-
ever, these high concentrations exceed the
physiological levels of THP in urine (20-200
µg/ml) and contradict results obtained by
Reinhart et al. (24), who observed a decrease
in the respiratory burst of PMNL after the
ingestion of E. coli F1+ bound to THP.
Our data agree with the results of Kuri-
yama and Silverblatt (8), who observed a
decrease in bacterial phagocytosis by PMNL,
even at THP concentrations <2.5 µg/ml.
Nevertheless, THP did not totally inhibit
bacterial binding and phagocytosis by mac-
rophages. Multiple mannose-sensitive bind-
ing sites between F1+ and THP have been
suggested to contribute to bacterial binding
(8). This could explain why THP only par-
tially blocked the phagocytosis of E. coli F1+
by mouse peritoneal macrophages since this
glycoprotein may act as a competitive inhib-
itor of E. coli F1+ binding to macrophage
surface receptors.
These results confirm other reports (24)
showing that THP inhibits binding between
type 1 fimbriated E. coli F1+ and PMN.
Kuriyama and Silverblatt (8) also observed
that type 1 fimbriated E. coli, which are
usually efficiently phagocytosed by PMN,
no longer become associated with these cells
if they are coated with THP. This antiopsonic
effect is similar to that observed when group
A streptococci bind fibrinogen to their sur-
face M protein (25).
The occurrence of THP on bladder or
kidney mucosa suggests a mechanism simi-
lar to the interaction of mucus with tracheal
and intestinal epithelium (26). There is still
no conclusive evidence that THP occurs in
917
Braz J Med Biol Res 34(7) 2001
Role of THP on phagocytosis of E. coli
transitional epithelium in vivo.
Our results support the view that the
soluble THP found in urine contains recep-
tors which are recognized by type 1 fim-
briae, thereby enhancing the urinary elimi-
nation of E. coli. However, in certain patho-
logical states, this glycoprotein may accu-
mulate on the renal parenchyma or peri-
renal tissue (27), and act as a binding site for
E. coli F1+, allowing bacterial colonization
and possibly decreasing phagocytosis by
macrophages.
Acknowledgments
We would like to thank Dr. Arício X.
Linhares (IB, Unicamp) for performing the
statistical analyses, Erivaldo José da Silva
for technical assistance, and Linda Gentry
El-Dash for revising the manuscript.
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Role Of Tamm-horsfall Protein In The Binding And In Vivo Phagocytosis Of Type 1 Fimbriated Escherichia Coli By Mouse Peritoneal Macrophages.

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Role Of Tamm-horsfall Protein In The Binding And In Vivo Phagocytosis Of Type 1 Fimbriated Escherichia Coli By Mouse Peritoneal Macrophages.

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