# Substance P Increases Cell-Surface Expression of CD74 (Receptor for Macrophage Migration Inhibitory Factor): In Vivo Biotinylation of Urothelial Cell-Surface Proteins

Authors: Katherine L. Meyer-Siegler; Shen-Ling Xia; Pedro L. Vera
Journal: Mediators of Inflammation
 (2009)

---

## Abstract

Macrophage migration inhibitory factor (MIF), an inflammatory cytokine, and its receptor CD74 are upregulated by bladder inflammation. MIF-mediated signal transduction involves binding to cell-surface CD74, this study documents, in vivo, MIF-CD74 interactions at the urothelial cell surface.
N-hydroxysulfosuccinimide biotin ester-labeled surface urothelial proteins in rats treated either with saline or substance P (SP, 40 μg/kg). The bladder was examined by histology and confocal microscopy. Biotinylated proteins were purified by avidin agarose, immunoprecipitated with anti-MIF or anti-CD74 antibodies, and detected with strepavidin-HRP. Only superficial urothelial cells were biotinylated. These cells contained a biotinylated MIF/CD74 cell-surface complex that was increased in SP-treated animals. SP treatment increased MIF and CD74 mRNA in urothelial cells. Our data indicate that intraluminal MIF, released from urothelial cells as a consequence of SP treatment, interacts with urothelial cell-surface CD74. These results document that our previously described MIF-CD74 interaction occurs at the urothelial cell surface.


---

## Body

## 1. Introduction

The
urothelium is no longer regarded as a passive barrier but rather as a complex
“sensory web” transducing signals from inside the viscus (and in response to
neuronal and local changes) through the activation of cell-surface receptors [1]. Therefore, the study of
interactions between urothelial cell-surface receptors expressed during normal
and diseased states and different molecules is likely to shed light on urothelial signal
transduction in health and disease.A central question in the study of tissue
response to inflammation is how cells titrate and integrate cytokine signals in
order to respond in an appropriate manner. Regulation of the specific pathway
through bioavailability ofligands and/or receptors is one potential
mechanism. Receptor bioavailability is achieved predominantly through the cell-surface
expression of specific functional receptors. The ligand-receptor interaction
results in the transduction of these extracellular signals across the plasma
membrane and, through the activation of intracellular signaling pathways,
affect the appropriate functional response.We have been studying
the role of a unique proinflammatory cytokine, macrophage migration inhibitory
factor (MIF) in experimental and clinical cystitis [2–4]. MIF is constitutively expressed and secreted
by numerous cell types, and significant levels can be found in blood, urine,
cerebrospinal and other body fluids [5, 6]. Therefore, circulating or extracellular MIF
is readily available, even under basal (“normal”) conditions [7]. The ubiquitous nature of this cytokine
suggests that cellular response to MIF is dictated either by increasing the
amount of available MIF or by changes in receptor bioavailability. We have
demonstrated that that subcutaneous SP increases intraluminal MIF amounts,
which is localized in preformed urothelial stores [4]. In addition, sequestering intraluminal MIF
with antibodies reduced SP-induced inflammatory changes in the rat bladder [8]. Thus, increased intraluminal MIF
participates in a feedback loop that continues to maintain SP-induced
inflammatory changes in the bladder. The precise mechanism of MIF action is
unknown. However, numerous studies have contributed to the current consensus
that the involvement of MIF is central in the initiation of the inflammatory response
[9, 10].Using in vitro models,
the MIF receptor was identified as the cell-surface form of CD74 (also known as
the invariant chain) [11]. The primary function of CD74 is to regulate
peptide loading onto major histocompatibility class II heterodimers [12]. However, a small portion of the total cell
CD74 content is expressed on cell surfaces, but the exact function of cell-surface
CD74 is not known [13]. Recent studies have documented the
interaction of MIF with CD74; however, it is not likely that this interaction
is sufficient to produce proinflammatory effects without the presence of CD44
(MIF-CD74 signaling component) [11, 14]. MIF, CD74, and CD44 are all upregulated
during experimental bladder inflammation suggesting that MIF-mediated signaling
may be involved in cystitis [15, 16]. Generally, cell-surface expression of CD74
is rather low, varying in monocytes from a few hundred to a few thousand
molecules per cell (for comparison, there are approx. 3 × 105 molecules of the MIF signal molecule CD44 per cell) again suggesting that cell-surface
availability of CD74 is the rate limiting variable in MIF signal transduction [17].We previously showed
that additional MIF is released from the bladder as part of the inflammatory
response, and is subsequently localized to umbrella cells in the urothelium [18]. In addition, we showed increased amounts of
CD74 protein localized to the urothelial layer suggesting that CD74 is a potential
receptor for MIF-mediated signal transduction during bladder inflammation [16]. Therefore, we hypothesize that urothelial
cell-surface CD74-MIF binding and subsequent association with CD44 activates
ERK1/2 signal transduction pathways. Taken together, our previous
results suggest that the bioavailability of the CD74 rather than MIF dictates
cell response. Therefore, we examined
the cell-surface location and extents of CD74 and MIF-CD74 in urothelial cells and the effects
of bladder inflammation induced by SP using in vivo biotinylation of
cell-surface proteins and confocal microscopy.


## 2. Methods

### 2.1. Animal Model

In vivo biotinylation of
urothelial cell-surface proteins—twelve male
Sprague-Dawley rats (250–300 gm) were
anesthetized with sodium pentobarbital (60 mg/kg; i.p.) and divided into two
groups: (1) saline (N = 6): bladders isolated from kidneys by cutting ureters;
bladders emptied of urine, rinsed twice with PBS, and replaced with 0.3 mL
biotinylation reagent (1 mg/mL N-hydroxysulfosuccinimide biotin ester, Pierce
Biochemicals, Rockford, Ill,
USA); saline treatment (s.c.); (2) substance P (SP, Sigma, St. Louis, Mo, USA;
N =  6): bladders
treated as in group (1) with SP treatment (40 μg/kg; s.c.). After 1 hour, the
bladders were excised and cut in half. Half of the bladder was placed in
formalin for histology and confocal microscopy, while the remaining piece was
carefully stretched and urothelial cells enriched using epithelial aggregate
separation and isolation (EASI) [19].


### 2.2. Histology

Biotinylation
of bladder tissue was detected by exposing frozen bladder sections (14μm
thick; intact bladders) to strepavidin- horseradish peroxidase (HRP) conjugate
and developing with 1%  3,3′-diaminobenzidine tetrahydrochloride in 0.3%
hydrogen peroxide (Sigma). Frozen sections (14 μm thick) of scraped bladders
were stained using hematoxylin to
document that only the epithelium was removed from the underlying tissue.


### 2.3. Purification and Detection of Biotinylated Proteins

Isolated urothelial cells were collected by
centrifugation at 10 000 g for 5 minutes, then washed in three times with 100 mM glycine in PBS pH 8.0 to inactivate residual biotinylation reagent. Half of
the collected urothelial cells were processed for protein and the remaining
used for RNA isolation.Proteins were extracted from
isolated urothelial cells by resuspension of washed cells in ice-cold RIPA
buffer (50 mM Tris-HCL, pH 7.5, 150 mM NaCl with 1% Triton X-100, 0.1% SDS, and
0.25% sodium deoxycholate, plus general protease inhibitors; Sigma), dounce
homogenization followed by centrifugation at 12 000 g for 15 minutes at 4°C.
Protein concentrations in the cleared cell lysates were determined using the
Micro-BCA protocol (Pierce
Biochemicals, Rockford, Ill,
USA).Urothelial
cell lysates (10μg total protein) were separated on 4–12% bis tris
acrylamide gels (NuPAGE, Invitrogen, Carlsbad, Calif, USA) and transferred to
PVDF. Western-blotting using antivimentin
(fibroblast marker, Neuromics, Edina, Minn, USA; #CH23010) verified that only
urothelial cells were removed and isolated. Western blots were stripped (0.2 M
glycine, pH 2.2 containing 0.1% SDS, 1% Tween-20) and reprobed with GAPDH
antibody (Santa Cruz Biotechnology, Santa Cruz, Calif, USA; sc-20357),
which served as a loading control.Urothelial
surface proteins labeled with biotin were purified from total urothelial cell
proteins by avidin-agarose affinity chromatography (A1979, Sigma). Nonexchangeable
biotin binding sites were blocked by incubating 100μL of avidin-agarose resin
with 400 μL of biotin (1 mg/mL) for 15 minutes at room temperature. Biotin was
eluted from exchangeable binding sites by washing the resin three times with
500 μL 0.1 M glycine, pH 2.0. The prepared resin was equilibrated with PBS and
incubated with 1 mg of total urothelial cell lysate. Biotinylated proteins were
eluted with 2 mM biotin and concentrated fivefold through 0.2 μm filters. Avidin-purified
proteins (15 μL of fivefold-concentrated avidin-agarose eluate) isolated from
each animal were separated by SDS-PAGE (Invitrogen) and transferred to PVDF. Blots
were blocked in blocking buffer (1% BSA, 10 mM PBS, pH 7.5. and 0.05% Tween-20)
for 1 hour at 37°C and total biotinylated protein detected by incubating
for 1 hour  at  37°C with HRP-labeled strepavidin (1:200 dilution
in blocking buffer R&D Systems, Minneapolis, Minn, USA).  The blot was then washed, and biotinylated
proteins visualized and quantified with a chemiluminescent imaging system (substrate,
Super Signal West Dura, Pierce; Kodak Image Station, Kodak, Rochester, NY, USA).
Total band intensities were determined using the manufacturer software (Kodak)
and expressed in arbitrary units. Data are expressed as median  ±
interquartile range.


### 2.4. Immunoprecipitation

Neither MIF nor CD74 could be
detected in the avidin-purified urothelial cell lysates using standard Western
blotting procedures, therefore immunoprecipitation was used. Equal amounts of
total protein form the urothelial cell lysates (1 mg, saline animals 4 and 5;
SP animals 9 and 10) were purified by avidin agarose, and the resulting biotinylated
proteins precleared by incubation with protein A agarose beads for 1 hour at 4°C.
The precleared biotinylated proteins were divided into two equal aliquots and
incubated overnight with 5 μg of the corresponding antibody (CD74, Santa Cruz
Biotechnology, sc-5438; or MIF, R&D Systems, AF-289-PB) at 4°C.
Protein A-agarose beads (50% slurry) were added to the mixture, which was
incubated for 1 hour at 4°C. Beads were then washed three times in
PBS and then resuspended in SDS sample buffer. Controls included incubation of
precleared samples with nonspecific goat IgG (Sigma), GAPDH antibody (Santa
Cruz Biotechnology, sc-20357), or samples without the addition of antibody in
the immunoprecipitation reaction. The resulting immunoprecipitated protein was
separated by 4–12% Bis-Tris
SDS-PAGE (Invitrogen) and transferred to PVDF. Incubating the blot overnight
with streptavidin-HRP conjugate (R&D Systems, 1:200 dilution in blocking
buffer, no detection or secondary antibody was used) was used to visualize only
immunoprecipitated biotinylated protein. Bands were visualized as described
above.


### 2.5. Coimmunoprecipitation

Cell-surface MIF-CD74 complexes
were isolated by incubating avidin-agarose purified biotinylated proteins
(purified from 1 mg total urothelial lysate) with MIF antibody (R&D
Systems, 1:1000 dilution, saline animals 1, 2, 3, and 6; SP animals 7, 8, 11,
and 12) at 4°C overnight. Immunoprecipitated proteins were
separated as described above, and CD74 containing MIF complexes were detected
by overnight incubation with anti-CD74 antibody (Santa Cruz Biotechnology,
sc-5438) followed by an antigoat HRP conjugate (Pierce) and visualized as
described above.


### 2.6. Pcr

Urothelial cell total RNA was isolated using TriZol reagent
(Invitrogen) with 1μg of RNA reverse transcribed and the resulting cDNA (2 μL)
used for relative quantitative PCR. Relative quantitative PCR conditions for MIF and CD74 were established by determining the linear range of the
target. Conditions for the 18S rRNA primer/competimer ratio were determined as
described by the manufacturer (3:7 primer:competimer ratio, Ambion, Austin, Tex,
USA). These primer rations optimized PCR conditions allow the generated 18S
rRNA product to be used as an internal standard to correct for variation in the
initial amount of total RNA reverse transcribed, as well as tube-to-tube
variations inherent in the PCR reaction. MIF and CD74 were amplified along with
the 18S rRNA internal standard using high stringency conditions: 30 cycles of
94°C 1 minute, 63°C 1 minute, 72°C 1 minute
as described previously [20]. PCR-generated
fragments were separated on precast 2% agarose gels containing ethidium bromide
(E-Gel, Invitrogen) and band intensity determined (Kodak, Rochester, NY, USA).
Relative band intensities were calculated by dividing total gene of interest
band intensity by 18S rRNA band intensity (internal standard), and fold change
in expression was determined by dividing SP-treated relative band intensity by
mean saline (control) relative band intensity. Data represent the mean  ±  SEM of
two separate PCR reactions per experimental animal. Reaction without reverse
transcriptase served as a negative control.


### 2.7. Confocal Microscopy

Frozen bladder sections (14μm; intact bladders) were exposed to
CD74 antiserum (sc-5438, specific for extracellular domain, Santa Cruz
Biotechnology) and visualized using secondary antiserum labeled with FITC or
TRITC. Control experiments included omission of the primary antiserum.Confocal microscopy for cell-surface CD74 was performed with a Zeiss LSM
510 laser scanning microscope (Thornwood, NY, USA) with Argon laser (for
excitation at 488 nm) and HeNe1 laser (for excitation at 543 nm). The slide
was mounted on a Zeiss-inverted microscope (Axiovert 100 M) with either
Plan-Neofluar 40×/0.75 N.A. air objective or Plan-Apochromat 100×/1.4 N.A.
oil objective. For each group of experiments, identical settings for the
imaging collection were used (via Reuse function). All the confocal
images were collected and analyzed using Zeiss software either provided with
the confocal imaging system or downloaded from Zeiss website (AxioVision LE, http://www.zeiss.com/C12567BE0045ACF1/Contents-Frame/3F3821B370EFC91CC125734C002FB38C).


## 2.1. Animal Model

In vivo biotinylation of
urothelial cell-surface proteins—twelve male
Sprague-Dawley rats (250–300 gm) were
anesthetized with sodium pentobarbital (60 mg/kg; i.p.) and divided into two
groups: (1) saline (N = 6): bladders isolated from kidneys by cutting ureters;
bladders emptied of urine, rinsed twice with PBS, and replaced with 0.3 mL
biotinylation reagent (1 mg/mL N-hydroxysulfosuccinimide biotin ester, Pierce
Biochemicals, Rockford, Ill,
USA); saline treatment (s.c.); (2) substance P (SP, Sigma, St. Louis, Mo, USA;
N =  6): bladders
treated as in group (1) with SP treatment (40 μg/kg; s.c.). After 1 hour, the
bladders were excised and cut in half. Half of the bladder was placed in
formalin for histology and confocal microscopy, while the remaining piece was
carefully stretched and urothelial cells enriched using epithelial aggregate
separation and isolation (EASI) [19].


## 2.2. Histology

Biotinylation
of bladder tissue was detected by exposing frozen bladder sections (14μm
thick; intact bladders) to strepavidin- horseradish peroxidase (HRP) conjugate
and developing with 1%  3,3′-diaminobenzidine tetrahydrochloride in 0.3%
hydrogen peroxide (Sigma). Frozen sections (14 μm thick) of scraped bladders
were stained using hematoxylin to
document that only the epithelium was removed from the underlying tissue.


## 2.3. Purification and Detection of Biotinylated Proteins

Isolated urothelial cells were collected by
centrifugation at 10 000 g for 5 minutes, then washed in three times with 100 mM glycine in PBS pH 8.0 to inactivate residual biotinylation reagent. Half of
the collected urothelial cells were processed for protein and the remaining
used for RNA isolation.Proteins were extracted from
isolated urothelial cells by resuspension of washed cells in ice-cold RIPA
buffer (50 mM Tris-HCL, pH 7.5, 150 mM NaCl with 1% Triton X-100, 0.1% SDS, and
0.25% sodium deoxycholate, plus general protease inhibitors; Sigma), dounce
homogenization followed by centrifugation at 12 000 g for 15 minutes at 4°C.
Protein concentrations in the cleared cell lysates were determined using the
Micro-BCA protocol (Pierce
Biochemicals, Rockford, Ill,
USA).Urothelial
cell lysates (10μg total protein) were separated on 4–12% bis tris
acrylamide gels (NuPAGE, Invitrogen, Carlsbad, Calif, USA) and transferred to
PVDF. Western-blotting using antivimentin
(fibroblast marker, Neuromics, Edina, Minn, USA; #CH23010) verified that only
urothelial cells were removed and isolated. Western blots were stripped (0.2 M
glycine, pH 2.2 containing 0.1% SDS, 1% Tween-20) and reprobed with GAPDH
antibody (Santa Cruz Biotechnology, Santa Cruz, Calif, USA; sc-20357),
which served as a loading control.Urothelial
surface proteins labeled with biotin were purified from total urothelial cell
proteins by avidin-agarose affinity chromatography (A1979, Sigma). Nonexchangeable
biotin binding sites were blocked by incubating 100μL of avidin-agarose resin
with 400 μL of biotin (1 mg/mL) for 15 minutes at room temperature. Biotin was
eluted from exchangeable binding sites by washing the resin three times with
500 μL 0.1 M glycine, pH 2.0. The prepared resin was equilibrated with PBS and
incubated with 1 mg of total urothelial cell lysate. Biotinylated proteins were
eluted with 2 mM biotin and concentrated fivefold through 0.2 μm filters. Avidin-purified
proteins (15 μL of fivefold-concentrated avidin-agarose eluate) isolated from
each animal were separated by SDS-PAGE (Invitrogen) and transferred to PVDF. Blots
were blocked in blocking buffer (1% BSA, 10 mM PBS, pH 7.5. and 0.05% Tween-20)
for 1 hour at 37°C and total biotinylated protein detected by incubating
for 1 hour  at  37°C with HRP-labeled strepavidin (1:200 dilution
in blocking buffer R&D Systems, Minneapolis, Minn, USA).  The blot was then washed, and biotinylated
proteins visualized and quantified with a chemiluminescent imaging system (substrate,
Super Signal West Dura, Pierce; Kodak Image Station, Kodak, Rochester, NY, USA).
Total band intensities were determined using the manufacturer software (Kodak)
and expressed in arbitrary units. Data are expressed as median  ±
interquartile range.


## 2.4. Immunoprecipitation

Neither MIF nor CD74 could be
detected in the avidin-purified urothelial cell lysates using standard Western
blotting procedures, therefore immunoprecipitation was used. Equal amounts of
total protein form the urothelial cell lysates (1 mg, saline animals 4 and 5;
SP animals 9 and 10) were purified by avidin agarose, and the resulting biotinylated
proteins precleared by incubation with protein A agarose beads for 1 hour at 4°C.
The precleared biotinylated proteins were divided into two equal aliquots and
incubated overnight with 5 μg of the corresponding antibody (CD74, Santa Cruz
Biotechnology, sc-5438; or MIF, R&D Systems, AF-289-PB) at 4°C.
Protein A-agarose beads (50% slurry) were added to the mixture, which was
incubated for 1 hour at 4°C. Beads were then washed three times in
PBS and then resuspended in SDS sample buffer. Controls included incubation of
precleared samples with nonspecific goat IgG (Sigma), GAPDH antibody (Santa
Cruz Biotechnology, sc-20357), or samples without the addition of antibody in
the immunoprecipitation reaction. The resulting immunoprecipitated protein was
separated by 4–12% Bis-Tris
SDS-PAGE (Invitrogen) and transferred to PVDF. Incubating the blot overnight
with streptavidin-HRP conjugate (R&D Systems, 1:200 dilution in blocking
buffer, no detection or secondary antibody was used) was used to visualize only
immunoprecipitated biotinylated protein. Bands were visualized as described
above.


## 2.5. Coimmunoprecipitation

Cell-surface MIF-CD74 complexes
were isolated by incubating avidin-agarose purified biotinylated proteins
(purified from 1 mg total urothelial lysate) with MIF antibody (R&D
Systems, 1:1000 dilution, saline animals 1, 2, 3, and 6; SP animals 7, 8, 11,
and 12) at 4°C overnight. Immunoprecipitated proteins were
separated as described above, and CD74 containing MIF complexes were detected
by overnight incubation with anti-CD74 antibody (Santa Cruz Biotechnology,
sc-5438) followed by an antigoat HRP conjugate (Pierce) and visualized as
described above.


## 2.6. Pcr

Urothelial cell total RNA was isolated using TriZol reagent
(Invitrogen) with 1μg of RNA reverse transcribed and the resulting cDNA (2 μL)
used for relative quantitative PCR. Relative quantitative PCR conditions for MIF and CD74 were established by determining the linear range of the
target. Conditions for the 18S rRNA primer/competimer ratio were determined as
described by the manufacturer (3:7 primer:competimer ratio, Ambion, Austin, Tex,
USA). These primer rations optimized PCR conditions allow the generated 18S
rRNA product to be used as an internal standard to correct for variation in the
initial amount of total RNA reverse transcribed, as well as tube-to-tube
variations inherent in the PCR reaction. MIF and CD74 were amplified along with
the 18S rRNA internal standard using high stringency conditions: 30 cycles of
94°C 1 minute, 63°C 1 minute, 72°C 1 minute
as described previously [20]. PCR-generated
fragments were separated on precast 2% agarose gels containing ethidium bromide
(E-Gel, Invitrogen) and band intensity determined (Kodak, Rochester, NY, USA).
Relative band intensities were calculated by dividing total gene of interest
band intensity by 18S rRNA band intensity (internal standard), and fold change
in expression was determined by dividing SP-treated relative band intensity by
mean saline (control) relative band intensity. Data represent the mean  ±  SEM of
two separate PCR reactions per experimental animal. Reaction without reverse
transcriptase served as a negative control.


## 2.7. Confocal Microscopy

Frozen bladder sections (14μm; intact bladders) were exposed to
CD74 antiserum (sc-5438, specific for extracellular domain, Santa Cruz
Biotechnology) and visualized using secondary antiserum labeled with FITC or
TRITC. Control experiments included omission of the primary antiserum.Confocal microscopy for cell-surface CD74 was performed with a Zeiss LSM
510 laser scanning microscope (Thornwood, NY, USA) with Argon laser (for
excitation at 488 nm) and HeNe1 laser (for excitation at 543 nm). The slide
was mounted on a Zeiss-inverted microscope (Axiovert 100 M) with either
Plan-Neofluar 40×/0.75 N.A. air objective or Plan-Apochromat 100×/1.4 N.A.
oil objective. For each group of experiments, identical settings for the
imaging collection were used (via Reuse function). All the confocal
images were collected and analyzed using Zeiss software either provided with
the confocal imaging system or downloaded from Zeiss website (AxioVision LE, http://www.zeiss.com/C12567BE0045ACF1/Contents-Frame/3F3821B370EFC91CC125734C002FB38C).


## 3. Results

### 3.1. in Vivo Biotinylation of Urothelial Cell Surface Proteins

We developed a
methodology to identify cell-surface protein expression in the urothelium using
in vivo biotinylation and also
determined urothelial cell-surface protein changes induced by SP treatment. In
both treatment and control bladders, the biotinylation reaction occurred
exclusively at the umbrella cell layer (Figures1(a)–1(d)) indicating
that only luminal cell-surface proteins were labeled with biotin.Bladder pieces remaining following
EASI were stained with hematoxylin (Figures 2(a)–2(d)). As seen in
both treatment and control bladders, our procedures resulted in the collection
of only urothelial cells with the lamina propria and smooth muscle tissue in
the bladder remaining intact (Figures 2(a)–2(d)). In addition, Western blotting of urothelial lysates for vimentin
(fibroblast marker) from all twelve animals was negative, while present in the
remaining (scraped) bladder pieces (Figures 2(e)), indicating that lamina propia
fibroblasts were not included in the isolated cells. Therefore, these results
suggest that our procedures successfully isolated urothelial cells without
contamination (as determined by histology and vimentin Western blotting) from
other cells in the lamina propia.In vivo biotinylation of urothelium. (a) and (c)
saline-treated bladders, (b) and (d) SP-treated bladders. Biotin labeling is
determined only with strepavidin-HRP and is localized to surface urothelium.
Calibration bar—(a), (b) = 400μm; (c), (d) =
100 μm.
(a)(b)(c)(d)Epithelial
aggregate separation and isolation (EASI) of rat bladder urothelium. Hematoxylin
staining of scraped bladders. (a) and (b) saline-treated bladders, (c) and (d)
SP-treated bladders. EASI only removes urothelium leaving intact lamina
propria. Calibration bar—(a), (c) =
400μm; (b), (d) = 100 μm. (e) Vimentin Western blot of total urothelial cell
lysates. Total protein from isolated urothelial cells was separated on 4–12% bis tris
acrylamide gels and transferred to
PVDF. Lanes 1–6 saline-treated
animals; lanes 7–12 SP-treated
animals, lane S—10 μg of scrapped
bladder protein (positive control). Vimentin Western blot was stripped and
reprobed with GAPDH which served as a loading control.
(a)(b)(c)(d)(e)


### 3.2. Sp-Induced Changes in Urothelial Cell-Surface Cd74 and Mif

Biotinylated proteins
from isolated urothelial cells were purified by avidin agarose and 15μL of the
fivefold concentrated eluate separated by reducing SDS-PAGE. The total amount
of cell-surface biotinylated protein from each was quantified by determining
total band intensity in each lane. Cell-surface protein as measured by
biotinylated band intensity increased tenfold (P = .002, Mann-Whitney)
following SP treatment (lanes 7–12, Figure 3(a);
total intensity 68340  ±  23710 arbitrary units) compared with control animals
(lanes 1–6, Figure 3(a);
total intensity 6600 ± 2725 arbitrary units).Expression of
CD74 and MIF on urothelial bladder surface. (a) Total biotinylated protein. Isolated
urothelial cells were lysed, biotinylated proteins were isolated by avidin
agarose affinity chromatography, separated by 4–12% bis tris
acrylamide gels electrophoresis,
transferred to PVDF and biotin containing protein bands visualized with
strepavidin-HRP only, no antibodies were used.
P: indicates precipitation of proteins with avidin agarose, D: indicates
detection with SA-HRP only, no antibodies were used. Lanes 1–6, saline-treated
animals; lanes 7–12 SP-treated
animals. (b) Immunoprecipitation of biotinylated CD74 or MIF. Isolated
urothelial cells were lysed, 1 mg of total biotinylated proteins was isolated by avidin
agarose affinity chromatography and biotinylated CD74 or MIF protein was
immunoprecipitated using appropriate antibodies. Precipitates were separated by
denaturing-reducing SDS PAGE and CD74 or MIF protein detected by
strepavidin-HRP. P: indicates precipitation of proteins with MIF antibody (left
panel) or CD74 antibody (right panel). D: indicates detection with SA-HRP only
(left and right panel), no antibodies were used. Lanes 1, 2 saline-treated
animals (number 4 and 5 from Figure3(a)), lanes 3, 4 SP-treated animals
(numbers 9 and 10 from Figure 3(a)). G: indicates immunoprecipitation with
GAPDH antibody documents that only biotinylated proteins were
immunoprecipitated, N: indicates immunoprecipitation with nonspecific goat IgG
documents antibody specificity. Lines and numbers to the far left indicate the
position of molecular weight markers. Asterisk indicates the location of 76 kDa band. Arrow indicates the location of 12 kDa uncomplexed MIF. (c) Coimmunoprecipitation
of cell-surface MIF/CD74 complexes. Isolated urothelial cells were lysed, 1 mg
total protein was used to purify biotinylated proteins by avidin agarose
affinity chromatography. Biotinylated MIF proteins were precipitated with an
anti-MIF antibody. Precipitates were separated by denaturing-reducing SDS PAGE
and CD74 protein containing bands identified using anti-CD74 antibody followed
by an antigoat-HRP. P: indicates precipitation of proteins by MIF antibody. D:
indicates detection with CD74 primary antibody and antigoat HRP secondary
antibody. Upper panel saline-treated animals, Lanes 1, 2, 3, 4, (numbers 1, 2,
3, and 6 from Figure 3(a)). Lower panel SP-treated animals, Lanes 1, 2, 3, 4,
(numbers 7, 8, 11, and 12 from Figure 3(a)). N: indicates immunoprecipitation
with nonspecific goat IgG documents antibody specificity.
(a)(b)(c)CD74
and MIF bands could not be detected by Western blotting of equal aliquots of
avidin-purified urothelial cell lysates (data not shown), suggesting that the
protein concentration was below the detection limit of this assay. Therefore,
to test the relationship between relative amounts of cell surface MIF/CD74 and
SP treatment, avidin-purified biotinylated proteins from urothelial lysates
were immunoprecipitated with MIF or CD74 antibodies and detected using HRP-labeled
strepavidin. SP treatment increased the amount of cell-surface CD74 as evidenced
by increased amount of immunoprecipitable biotinylated CD74 from SP-treated
urothelial cells (P = .03, Mann-Whitney; right panel, lanes 1, 2, animals
4, 5; lanes 3, 4, animals 9, 10; Figure 3(b)). In addition, the amount of cell
surface (biotinylated) immunoprecipitable MIF was increased with SP treatment (P = .03,
Mann-Whitney; left panel, lanes 1, 2, animals 4, 5; lanes 3, 4, animals 9, 10; Figure
3(b)). Not all the MIF was complexed to the
76 kDa band since 12 kDa MIF was
found in the MIF immunoprecipitation (Figure 3(b), left panel, arrow).
Immunoprecipitation with GAPDH was used to confirm that only biotinylated
proteins were present as indicated by the absence of SA-HRP reactive bands (Figure
3(b), lane G, left and right panels).
Goat IgG was used as a control of antibody specificity (Figure 3(b), lane N).Both CD74 and MIF immunoprecipitation reactions showed a biotinylated
(cell-surface) protein band at the same molecular weight (76 kDa), which could
be attributed to a cell-surface CD74-MIF complex that is not broken upon
reducing conditions (asterisk Figure3(b)). Therefore, co-immunoprecipitation
of cell-surface proteins with MIF antibody followed by CD74 Western blotting
was used to determine if CD74 was associated with immunoprecipitable
biotinylated (cell-surface) MIF. In addition co-immunoprecipitation was used to
determine if SP treatment increased the amount of this complex. SP treatment
increased the amount of this complex greater than twofold (P = .02, Figure
3(c) saline lanes 1–4, animals 1, 2,
3, 6, total intensity 32733  ±  17311 arbitrary units; SP lanes 1–4, animals 7, 8,
11, 12, total intensity 13956 ± 2505 arbitrary units). These results suggest
that SP treatment increased availability of cell-surface CD74 receptor for MIF
binding.Along with increased cell-surface CD74 and MIF SP-treatment also resulted
in increased gene expression of both CD74  and MIF within isolated urothelial
cells (Figure4).(a) MIF and CD74
mRNA expressions in
isolated urothelial cells. Total RNA was isolated from urothelial cells and CD74
or MIF-specific primers used to amplify mRNA by endpoint PCR. A CD74 and MIF
mRNA is increased in isolated urothelial cells after SP treatment. Data are
representative of 5 salines and 5 SP-treated animals. 18S rRNA was used as a
control for the amount of total RNA added to each reaction tube. (b) Relative
expression of CD74 and MIF in isolated urothelial cells. Data are expressed as
fold change calculated by determining mean gene expression ratio (net intensity
of gene-specific band divided by net intensity of 18S rRNA band) normalized to
saline controls. (P***<.001, n = 5).
(a)(b)Finally, in order to confirm that CD74 localizes to the bladder
urothelial cell surface and that cell-surface expression is increased with SP
treatment, nonpermeabilized bladder tissue was immunostained using CD74
antibody (Santa Cruz, sc-5438) and examined by confocal microscopy. CD74 antibody
detected expression in a subpopulation of these cells. As seen in Figure5, CD74 protein expression is localized to the cell surface and is
increased with SP treatment (Figures 5(b), 5(c)). Control experiments included
tissue not exposed to secondary antibody (Figure 5(d)).CD74 confocal
microscopy. Frozen
bladder sections (14μm; intact bladders) were exposed to CD74 antiserum and
visualized using secondary antiserum labeled with FITC or TRITC. Control
experiments included omission of the primary antiserum (panel D). (a) Saline-treated
bladder with 40× objective. (b) SP-treated bladder with 40× objective. (c)
SP-treated bladder with 100× objective. (d)
Saline-treated bladder with 40× objective, secondary antibody only. Excitation wavelength of 488 nm
wasused and emission wavelength of 505 nm and up was collected.
Frame size of the image for (a), (b), and (d) is 230.3 μm × 230.3 μm;
optical thickness of the images is 2 μm. Frame size of the image for (c) is
92.1 μm × 92.1 μm; optical thickness of the image is 0.9 μm. LP: lamina propria; NUC: nucleus;
arrows indicate luminal surface of urothelial cells.
(a)(b)(c)(d)


## 3.1. in Vivo Biotinylation of Urothelial Cell Surface Proteins

We developed a
methodology to identify cell-surface protein expression in the urothelium using
in vivo biotinylation and also
determined urothelial cell-surface protein changes induced by SP treatment. In
both treatment and control bladders, the biotinylation reaction occurred
exclusively at the umbrella cell layer (Figures1(a)–1(d)) indicating
that only luminal cell-surface proteins were labeled with biotin.Bladder pieces remaining following
EASI were stained with hematoxylin (Figures 2(a)–2(d)). As seen in
both treatment and control bladders, our procedures resulted in the collection
of only urothelial cells with the lamina propria and smooth muscle tissue in
the bladder remaining intact (Figures 2(a)–2(d)). In addition, Western blotting of urothelial lysates for vimentin
(fibroblast marker) from all twelve animals was negative, while present in the
remaining (scraped) bladder pieces (Figures 2(e)), indicating that lamina propia
fibroblasts were not included in the isolated cells. Therefore, these results
suggest that our procedures successfully isolated urothelial cells without
contamination (as determined by histology and vimentin Western blotting) from
other cells in the lamina propia.In vivo biotinylation of urothelium. (a) and (c)
saline-treated bladders, (b) and (d) SP-treated bladders. Biotin labeling is
determined only with strepavidin-HRP and is localized to surface urothelium.
Calibration bar—(a), (b) = 400μm; (c), (d) =
100 μm.
(a)(b)(c)(d)Epithelial
aggregate separation and isolation (EASI) of rat bladder urothelium. Hematoxylin
staining of scraped bladders. (a) and (b) saline-treated bladders, (c) and (d)
SP-treated bladders. EASI only removes urothelium leaving intact lamina
propria. Calibration bar—(a), (c) =
400μm; (b), (d) = 100 μm. (e) Vimentin Western blot of total urothelial cell
lysates. Total protein from isolated urothelial cells was separated on 4–12% bis tris
acrylamide gels and transferred to
PVDF. Lanes 1–6 saline-treated
animals; lanes 7–12 SP-treated
animals, lane S—10 μg of scrapped
bladder protein (positive control). Vimentin Western blot was stripped and
reprobed with GAPDH which served as a loading control.
(a)(b)(c)(d)(e)


## 3.2. Sp-Induced Changes in Urothelial Cell-Surface Cd74 and Mif

Biotinylated proteins
from isolated urothelial cells were purified by avidin agarose and 15μL of the
fivefold concentrated eluate separated by reducing SDS-PAGE. The total amount
of cell-surface biotinylated protein from each was quantified by determining
total band intensity in each lane. Cell-surface protein as measured by
biotinylated band intensity increased tenfold (P = .002, Mann-Whitney)
following SP treatment (lanes 7–12, Figure 3(a);
total intensity 68340  ±  23710 arbitrary units) compared with control animals
(lanes 1–6, Figure 3(a);
total intensity 6600 ± 2725 arbitrary units).Expression of
CD74 and MIF on urothelial bladder surface. (a) Total biotinylated protein. Isolated
urothelial cells were lysed, biotinylated proteins were isolated by avidin
agarose affinity chromatography, separated by 4–12% bis tris
acrylamide gels electrophoresis,
transferred to PVDF and biotin containing protein bands visualized with
strepavidin-HRP only, no antibodies were used.
P: indicates precipitation of proteins with avidin agarose, D: indicates
detection with SA-HRP only, no antibodies were used. Lanes 1–6, saline-treated
animals; lanes 7–12 SP-treated
animals. (b) Immunoprecipitation of biotinylated CD74 or MIF. Isolated
urothelial cells were lysed, 1 mg of total biotinylated proteins was isolated by avidin
agarose affinity chromatography and biotinylated CD74 or MIF protein was
immunoprecipitated using appropriate antibodies. Precipitates were separated by
denaturing-reducing SDS PAGE and CD74 or MIF protein detected by
strepavidin-HRP. P: indicates precipitation of proteins with MIF antibody (left
panel) or CD74 antibody (right panel). D: indicates detection with SA-HRP only
(left and right panel), no antibodies were used. Lanes 1, 2 saline-treated
animals (number 4 and 5 from Figure3(a)), lanes 3, 4 SP-treated animals
(numbers 9 and 10 from Figure 3(a)). G: indicates immunoprecipitation with
GAPDH antibody documents that only biotinylated proteins were
immunoprecipitated, N: indicates immunoprecipitation with nonspecific goat IgG
documents antibody specificity. Lines and numbers to the far left indicate the
position of molecular weight markers. Asterisk indicates the location of 76 kDa band. Arrow indicates the location of 12 kDa uncomplexed MIF. (c) Coimmunoprecipitation
of cell-surface MIF/CD74 complexes. Isolated urothelial cells were lysed, 1 mg
total protein was used to purify biotinylated proteins by avidin agarose
affinity chromatography. Biotinylated MIF proteins were precipitated with an
anti-MIF antibody. Precipitates were separated by denaturing-reducing SDS PAGE
and CD74 protein containing bands identified using anti-CD74 antibody followed
by an antigoat-HRP. P: indicates precipitation of proteins by MIF antibody. D:
indicates detection with CD74 primary antibody and antigoat HRP secondary
antibody. Upper panel saline-treated animals, Lanes 1, 2, 3, 4, (numbers 1, 2,
3, and 6 from Figure 3(a)). Lower panel SP-treated animals, Lanes 1, 2, 3, 4,
(numbers 7, 8, 11, and 12 from Figure 3(a)). N: indicates immunoprecipitation
with nonspecific goat IgG documents antibody specificity.
(a)(b)(c)CD74
and MIF bands could not be detected by Western blotting of equal aliquots of
avidin-purified urothelial cell lysates (data not shown), suggesting that the
protein concentration was below the detection limit of this assay. Therefore,
to test the relationship between relative amounts of cell surface MIF/CD74 and
SP treatment, avidin-purified biotinylated proteins from urothelial lysates
were immunoprecipitated with MIF or CD74 antibodies and detected using HRP-labeled
strepavidin. SP treatment increased the amount of cell-surface CD74 as evidenced
by increased amount of immunoprecipitable biotinylated CD74 from SP-treated
urothelial cells (P = .03, Mann-Whitney; right panel, lanes 1, 2, animals
4, 5; lanes 3, 4, animals 9, 10; Figure 3(b)). In addition, the amount of cell
surface (biotinylated) immunoprecipitable MIF was increased with SP treatment (P = .03,
Mann-Whitney; left panel, lanes 1, 2, animals 4, 5; lanes 3, 4, animals 9, 10; Figure
3(b)). Not all the MIF was complexed to the
76 kDa band since 12 kDa MIF was
found in the MIF immunoprecipitation (Figure 3(b), left panel, arrow).
Immunoprecipitation with GAPDH was used to confirm that only biotinylated
proteins were present as indicated by the absence of SA-HRP reactive bands (Figure
3(b), lane G, left and right panels).
Goat IgG was used as a control of antibody specificity (Figure 3(b), lane N).Both CD74 and MIF immunoprecipitation reactions showed a biotinylated
(cell-surface) protein band at the same molecular weight (76 kDa), which could
be attributed to a cell-surface CD74-MIF complex that is not broken upon
reducing conditions (asterisk Figure3(b)). Therefore, co-immunoprecipitation
of cell-surface proteins with MIF antibody followed by CD74 Western blotting
was used to determine if CD74 was associated with immunoprecipitable
biotinylated (cell-surface) MIF. In addition co-immunoprecipitation was used to
determine if SP treatment increased the amount of this complex. SP treatment
increased the amount of this complex greater than twofold (P = .02, Figure
3(c) saline lanes 1–4, animals 1, 2,
3, 6, total intensity 32733  ±  17311 arbitrary units; SP lanes 1–4, animals 7, 8,
11, 12, total intensity 13956 ± 2505 arbitrary units). These results suggest
that SP treatment increased availability of cell-surface CD74 receptor for MIF
binding.Along with increased cell-surface CD74 and MIF SP-treatment also resulted
in increased gene expression of both CD74  and MIF within isolated urothelial
cells (Figure4).(a) MIF and CD74
mRNA expressions in
isolated urothelial cells. Total RNA was isolated from urothelial cells and CD74
or MIF-specific primers used to amplify mRNA by endpoint PCR. A CD74 and MIF
mRNA is increased in isolated urothelial cells after SP treatment. Data are
representative of 5 salines and 5 SP-treated animals. 18S rRNA was used as a
control for the amount of total RNA added to each reaction tube. (b) Relative
expression of CD74 and MIF in isolated urothelial cells. Data are expressed as
fold change calculated by determining mean gene expression ratio (net intensity
of gene-specific band divided by net intensity of 18S rRNA band) normalized to
saline controls. (P***<.001, n = 5).
(a)(b)Finally, in order to confirm that CD74 localizes to the bladder
urothelial cell surface and that cell-surface expression is increased with SP
treatment, nonpermeabilized bladder tissue was immunostained using CD74
antibody (Santa Cruz, sc-5438) and examined by confocal microscopy. CD74 antibody
detected expression in a subpopulation of these cells. As seen in Figure5, CD74 protein expression is localized to the cell surface and is
increased with SP treatment (Figures 5(b), 5(c)). Control experiments included
tissue not exposed to secondary antibody (Figure 5(d)).CD74 confocal
microscopy. Frozen
bladder sections (14μm; intact bladders) were exposed to CD74 antiserum and
visualized using secondary antiserum labeled with FITC or TRITC. Control
experiments included omission of the primary antiserum (panel D). (a) Saline-treated
bladder with 40× objective. (b) SP-treated bladder with 40× objective. (c)
SP-treated bladder with 100× objective. (d)
Saline-treated bladder with 40× objective, secondary antibody only. Excitation wavelength of 488 nm
wasused and emission wavelength of 505 nm and up was collected.
Frame size of the image for (a), (b), and (d) is 230.3 μm × 230.3 μm;
optical thickness of the images is 2 μm. Frame size of the image for (c) is
92.1 μm × 92.1 μm; optical thickness of the image is 0.9 μm. LP: lamina propria; NUC: nucleus;
arrows indicate luminal surface of urothelial cells.
(a)(b)(c)(d)


## 4. Discussion

MIF is a
constitutively expressed, multifunctional cytokine that functions to counterregulate
the effects of glucocorticoids and to stimulate the synthesis and secretion of
other proinflammatory mediators such as TNF-α and IL-1β, as such it has been
suggested that MIF is a key initiator of the inflammatory cascade [21, 22].
In addition to its function in the inflammatory response, MIF functions as an
autocrine regulator of cell proliferation and differentiation. Serum-starved
NIH/3T3 cells secreted endogenous MIF as a consequence of serum stimulation
resulting in proliferation of quiescent cells. This response was associated
with phosphorylation and subsequent activation of ERK 1/2 [23].SP is an afferent
nerve fiber neurotransmitter that mediates inflammation by inducing
vasodilatation and plasma extravasation [24]. SP has a short half-life
suggesting that inflammation initially induced by SP is maintained by the
release of other proinflammatory mediators [24]. Our previous experiments
established MIF as one such SP-induced proinflammatory mediator [4]. SP affects the rat bladder
by upregulating MIF expression and inducing
the release of urothelial MIF into the bladder lumen [4]. Sequestering the MIF
released into the bladder lumen with anti-MIF antibodies decreases SP-induced
inflammatory changes in the bladder [8]. In addition, our studies
have shown that the MIF receptor, CD74, is also found in the urothelium and
that SP increases CD74 protein and mRNA amounts in the bladder [15]. The findings of this study
show that this increase in MIF and CD74 mRNA is localized to the urothelium.
Thus, we propose that MIF, constitutively expressed by the urothelium and
upregulated in SP-induced inflammation, binds to cell-surface CD74 receptor,
which perpetuates inflammation. Direct evidence of SP-induced MIF-CD74
interaction had not been described previously. The aim of the present
experiments was to determine SP-induced cell-surface CD74 expression and the
interaction of cell-surface CD74 with MIF in the rat urothelium.It is difficult to account for the absence of
an MIF response under “normal” physiological conditions considering the
ubiquitous expression of this protein [25]. MIF is readily detected in urine suggesting
that bioactive MIF is readily available in the local environment [2, 4]. CD74 amounts within the normal bladder are
present in low amounts and our previous immunoprecipitation studies demonstrated
increased MIF-CD74 complexes as a result of SP-induced inflammation, suggesting
that it is likely CD74 availability controls the cellular response to exogenous
MIF [15]. Interestingly, we found
that CD74 expression in SP-treated bladder is limited largely to the urothelial
cell layer surface, a differentiated cell type believed toplay a
role in maintaining the integrity of the urothelium (Figure 5).Under normal physiological conditions,
urothelial cells that do not express cell-surface CD74 (or express small
amounts of CD74) would not be able to respond to secreted MIF via the
well-described CD74-mediated ERK-activation. The results presented here suggest
that increased cell-surface expression of CD74 induced by SP results in greater
binding of secreted MIF molecules by CD74, and therefore greater activation of
CD74-mediated signaling pathways (e.g., ERK-activation). In particular, the
results of the immunoprecipitation experiments described here establish that SP
treatment results in an increased cell-surface CD74/MIF complex when compared
with saline controls (Figure3(c)). Stable immunoprecipitable CD74 complexes of
similar molecular weights to what we report here have been identified
previously in both human peripheral blood T-cells and duodenal epithelial cells
[26, 27]. The exact nature and function of these
complexes was not established.In their entirety,
these results suggest that the relative contributions of MIF to normal bladder
function are minimal, but inflammatory processes are reliant upon bioactive MIF
and cell-surface expression of the MIF receptor, CD74. This would imply that
pharmacologic inhibition of MIF (e.g., using pharmacological antagonists of MIF
tautomerase activity with ISO-1) would have little effect on normal bladder
function, but may prove to be a potent bladder inflammatory inhibitor. As
evidenced by the data presented here, a model for MIF response at the cellular
level emerges: under normal conditions although MIF is present in the
surrounding tissues as well as in the peripheral blood; the cell-surface
receptor is absent. In response to inflammatory stimuli, epithelial cells
express the MIF receptor (CD74) on their surface, thus initiating inflammatory
response.


## 5. Conclusions

In
summary, the present study documents increased MIF and urothelial cell-surface
CD74 expression during inflammation. The emerging role of MIF in bladder
inflammation suggests that modulating this cytokine activity may result in new,
selective, and therapeutic modalities.



---

*Source: 535348-2009-03-22.xml*