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-Defensins HNPs-1, -2, and -3 in Renal Cell Carcinoma
From Department II,* Section of TransplantationImmunology and Immunohematology, Medical University Clinic, and theMedical and Natural Sciences Research Center,and the Children’s Hospital,¶ Eberhard-Karls-University, Tübingen, Germany; the Department ofNephrology and Rheumatology, Center ofInternal Medicine, Georg-August-University, Göttingen, Germany;and the Medical Faculty, Institute ofPathology, University of Belgrade, Belgrade, Yugoslavia
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Abstract |
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-defensins human neutrophil peptides (HNPs)-1,
-2, and -3 have been described as cytotoxic peptides with
restricted expression in neutrophils and in some lymphocytes. In this
study we report that HNPs-1, -2, and -3 are also
expressed in renal cell carcinomas (RCCs). Several RCC lines were found
to express mRNA as well as the specific peptides of HNP-1,
-2, and -3 demonstrated by reverse transcriptase-polymerase
chain reaction, mass spectrometric, and flow cytometric
analyses. At physiological concentrations HNPs-1, -2,
and -3 stimulated cell proliferation of selected RCC lines in
vitro but at high concentrations were cytotoxic for all RCC
lines tested. As in RCC lines, -defensins were also detected
in vivo in malignant epithelial cells of 31 RCC tissues
in addition to their expected presence in neutrophils. In most RCC
cases randomly, patchy immunostaining of -defensins on
epithelial cells surrounding neutrophils was seen, but in six
tumors of higher grade malignancy all tumor cells were diffusely
stained. Cellular necrosis observed in RCC tissues in association with
extensive patches of HNP-1, -2, and -3, seemed
to be related to high concentrations of -defensins. The in
vitro and in vivo findings suggest that
-defensins are frequent peptide constituents of malignant epithelial
cells in RCC with a possible direct influence on tumor
proliferation.
To date, two classes of human defensins, termed "-defensins" and
"ß-defensins," have been identified that differ with respect to
their localization and linkage of cysteine residues, precursor peptide
structure, and pattern of tissue expression. Whereas ß-defensins are
most abundant in epithelial cells of the lung,11,12
skin,13
and urogenital tract,14
the
-defensins were first found in human polymorphonuclear leukocytes
(PMNs) and intestinal Paneth cells.15,16
Human neutrophil
peptides (HNPs)-1, -2, and -3, and to a lesser extent HNP-4, are major
constituents of the dense azurophilic granules of neutrophils, from
which they are discharged into phagolysosomes on ingestion of
microbes.17
Within these compartments they are able to
form pores that disrupt ion fluxes and eventuate in lysis of the
engulfed microorganisms without major damage to host tissues. Two other
-defensins, HD-5 and HD-6, are sequestered in lysozyme-rich granules
of the Paneth cells and are also thought to support local antimicrobial
defense mechanisms in the gut.18
Although normally
sequestered in PMN and Paneth cells, -defensins have also been
recently described in the walls of coronary vessels.19
Furthermore HNPs-1, -2, and -3 were found to be expressed in specific
lymphocyte and monocyte subpopulations.20
In addition to their role in host defense against microbial infection,
defensins are thought to contribute to tissue inflammation and repair.
HNPs-1, -2, and -3 display various proinflammatory activities such as
the induction of histamine release from mast cells21
and
chemotactic effects for human monocytes,22
dendritic
cells,23
and PMNs as well as T cells.24
-Defensins have been also reported to exert mitogenic effects on
murine fibroblasts and on epithelial cells with possible consequences
on tissue repair.25
They modulate binding and catabolism
of low-density lipoproteins by vascular endothelial cells and therefore
have been suggested to be involved in providing a link between
inflammation and atherosclerosis.26
Furthermore,
-defensins were shown in mice to activate T-helper cell responses
in vitro and enhance systemic antigen-specific IgG
production in vivo. Thus, they were suggested to provide
signals for linking innate and acquired immunity.27
In a
recent study, -defensins were found to represent dominant
HLA-DR-binding peptides on normal and malignant hematopoietic
progenitor cells suggesting major histocompatibility complex blockade
for antigen presentation.28
Although defensins today seem to have diverse functional activities in
innate antimicrobial immunity, a few reports also indicated the
presence of defensins in tissues of epithelial
tumors.29-31
In this study, -defensins are for the
first time shown to be synthesized by renal cell carcinomas (RCCs) and
to influence proliferation of a subset of renal malignant cells
in vitro. In addition, endogenous binding of -defensins
to HLA-class II molecules in RCC was found suggesting a blockade of
antigen-presenting function, as in myeloma and hematopoietic progenitor
cells.28
Thus, -defensins could belong to tumor
substrates that modulate malignant cell growth and enhance immune
escape in RCC.
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Materials and Methods |
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In this study, eight RCC lines were used that included five lines (A-498, Caki-2, 786-0, 769-P, ACHN) purchased from the American Type Culture Collection (ATCC; Manassas, VA), one line (LE 9211-RCC) obtained from Dr. B. van den Eynde (Ludwig Cancer Institute, Brussels, Belgium) and two lines (TW33, N43) were established from renal tumor tissues in our laboratory according to the previously described method.32 All cell lines represented tumor cells derived from typical clear cell, chromophilic/papillary, and chromophobe types of RCC. Immunohistochemical analyses showed a pure population of cytokeratin-positive cells in all cultures of these cell lines. In addition, an Epstein-Barr virus-transformed B-cell line Cox, as well as the promyelocytic cell line HL-60 also purchased from ATCC were used for comparison. Isolation, culture, and characterization of primary tubular epithelial cells, TK163, have been described previously.33
The RCC lines were routinely grown as monolayers and the cell lines Cox and HL-60 as suspension cultures in RPMI 1640 (Life Technology GmbH, Karlsruhe, Germany) supplemented with 10% fetal calf serum, penicillin, and streptomycin (standard culture medium) at 37°C in an atmosphere with 5% CO2. All RCC lines were detached from culture flasks with trypsin/ethylenediaminetetraacetic acid (EDTA) (Life Technology GmbH, Karlsruhe, Germany) for fluorescence-activated cellsorting (FACS) and polymerase chain reaction (PCR) analysis.
Tissue Sampling
RCC tumor specimens were obtained from 31 patients undergoing nephrectomy. Five biopsies were derived from kidneys of healthy organ donors that were not transplanted because of vascular abnormalities. A small portion of each sample was submitted to conventional histopathological processing and light microscopic evaluation. The other parts of the RCC specimens and normal kidney tissues were put into cell culture medium RPMI 1640 (Life Technology GmbH) immediately after removal, snap-frozen, and stored in liquid nitrogen until further analysis.
Antibodies
For analysis of HNP-1, -2, and -3 expression, the murine
monoclonal antibody (mAb) DEF-3 (Bachem Biochemica, Heidelberg,
Germany), as well as another murine mAb prepared against highly
purified HNP-1 (kindly provided by Dr. T. Ganz, Department of
Pulmonary/Critical Care Medicine, School of Medicine, University of
California, Los Angeles, CA)34
were applied. Both
antibodies also recognize HNPs-2 and -3, but not HNP-4. For
fluorescence double staining a polyclonal anti-human defensin antibody
was produced by immunizing rabbits with human -defensins isolated
from leukapheresis products and conjugated to keyhole limpet
hemocyanin. IgG fractions of the immunized rabbit sera were separated
on a protein A column. Specificity of the antibody was controlled by
enzyme-linked immunosorbent assay with the HNP-1, -2, and -3 isolates
as well as by comparative immunohistochemical staining with the mAbs
against defensin.
In addition, mAb clone NP57 against human neutrophil elastase and clone 35H11 against cytokeratin 8 were obtained from DAKO (Hamburg, Germany). The mAb W6/32.HL directed against HLA-A, -B, and -C antigens and the mAb W6/32.HK,35 an inactive variant of W6/32.HLK, as well as the mAb L243 with specificity for HLA-DR molecules were purchased from the ATCC.
Immunohistochemistry
Five-µm-thick frozen sections cut from each tumor specimen were fixed in acetone for 10 minutes at -20°C and used for indirect immunoperoxidase staining. mAbs against defensin (diluted 1:500) and neutrophil elastase (diluted 1:250) were used as primary antibodies and goat anti-mouse EnVision horseradish-peroxidase conjugate (DAKO, Hamburg, Germany) as the second layer and detection system for antibody binding. All experiments included sections stained with undiluted supernatant of the mAb W6/32.HL as positive and of the mAb W6/32.HK as negative control to exclude nonspecific staining. All sections were counterstained with hemalaun (Merck, Darmstadt, Germany) and evaluated under a Zeiss light microscope (Zeiss, Jena, Germany).
Triple Immunofluorescence Labeling
After fixation the slides were incubated for 1 hour at room
temperature with mAb against cytokeratin 8 (diluted 1:25), followed by
a fluorescein isothiocyanate-conjugated goat anti-mouse antibody
(diluted 1:200; Dianova, Hamburg, Germany). Then, the polyclonal
antibody against -defensins (diluted 1:100) was added followed by
Cy3-conjugated goat anti-rabbit antibody (diluted 1:2000; Dianova,
Hamburg, Germany). The cell nuclei were identified by counterstaining
with 4,6-diamino-2-phenylindolyl-dihydrochloride (DAPI; 1 µg/ml).
Negative controls were performed in all experiments by omitting the
first antibodies. The slides were visualized by epifluorescence light
microscopy (Olympus BX-60, Hamburg, Germany). Digital pictures from
every fluorescence channel were taken and super imposed for the
specific antibody stains as well as for each negative control labeling
using the software DOKU from Soft Imaging Systems
(Leinfelden-Echterdingen, Germany).
Flow Cytometry and Confocal Microscopy
Intracellular expression of HNP-1, -2, and -3 was assessed in RCC lines after thawing and culturing for 72 hours. For FACS analysis, the RCC lines were detached from the cell-culture flask by trypsin/EDTA, placed on ice, and washed twice with washing buffer [0.1% w/v bovine serum albumin in phosphate-buffered saline (PBS)]. For confocal microscopy, the RCC lines were directly cultured on round coverslips in 12-well plates (Greiner, Frickenhausen, Germany) for 12 hours. After washing with PBS, RCCs cultured on slides were fixed with 4% paraformaldehyde for 10 minutes at room temperature.
To block nonspecific labeling, cells were first incubated for 10 minutes with human standard immunoglobulin (Polyglobin N; Bayer AG, Leverkusen, Germany). After two further washes with washing buffer the RCC lines were fixed and permeabilized with the Fix and Perm kit (An der Grub, Kaumberg, Austria) according to the manufacturer’s instructions. Thereafter, the cells were labeled with the mAbs against HNPs-1, -2, and -3 as primary antibody, and fluorescein isothiocyanate or Cy2-conjugated goat anti-mouse IgG (Dianova) as the secondary antibody layer. Fluorescence of the cells in suspension was evaluated on a FACScan (Becton Dickinson, Mountain View, CA) using the WinMDI flow-cytometry software version 2.5 (http://facs.scripps.edu/). Fluorescence of the cells cultured on the slides was evaluated with a LSM410 LaserScan microscope (Zeiss) using a 488-nm argon laser for excitation.
All experiments included labeling with the negative control mAb W6/32.HK and the positive control mAb W6/32.HL. The promyelocytic leukemia cell line HL-60 was used as a positive control cell line for intracellular expression of HNPs-1, -2, and -3.
Reverse-Transcriptase (RT)-PCR, Cycle-Sequencing of PCR Products
Total RNA was prepared from RCC lines using the RNeasy-Kit (Qiagen Ltd., Hilden, Germany). After DNA digestion with DNase I (Roche Molecular Biochemicals, Mannheim, Germany) for 20 minutes at 25°C, 1 µg of total RNA was reverse-transcribed with 0.5 µg of oligo (dT)12-18 using the Advantage RT-for-PCR kit (Clontech, Heidelberg, Germany). For amplification of HNP-1, -2, and -3 cDNA, primer 1 (5'-CACTCCAGGCAAGAGCTGATGAGGT-TG-3', position 2688 to 2714) and primer 2 (5'-AATGCCCAGAGTCTTCCCTGGTAGATG-3' position 3454 to 3482)12,36,37 were synthesized on a LKB Gene Assembler Plus (Amersham Pharmacia Biotech, Freiburg, Germany) and purified with NAP 10 columns (Amersham Pharmacia Biotech). As an internal control in all tests, ß-actin was amplified (product of 317 bp) using primer A (5'-TCAGAAGGATTCCTATGTGGGC-3') and primer B (3'-CCATCA CGATGCCAG-TGGTA-5'). PCR amplifications were performed in a 50-µl reaction mixture of 250 ng cDNA, 5 µl GeneAmp10x PCR buffer (Applied Biosystems, Weiterstadt, Germany), 5% dimethyl sulfoxide, 200 µmol/L of each dNTP, 1 U AmpliTaq DNA Polymerase (Applied Biosystems, Weiterstradt, Germany), and 100 ng of each primer. Amplification was performed in a Primus 25 PCR System (MWG-Biotech, Ebersberg, Germany) starting with an initial denaturation step at 95°C for 7 minutes for 26 PCR cycles, each of which consisted of denaturation at 94°C for 15 seconds, annealing at 68°C for 45 seconds, and extension at 72°C for 30 seconds were followed by 20 cycles of denaturation at 95°C for 15 seconds, annealing at 62°C for 45 seconds, and extension at 72°C for 30 seconds. The last cycle was terminated by a final extension at 72°C for 10 minutes. Each amplification product was run on a 2% agarose gel and stained with ethidium bromide to monitor for specificity before direct cycle sequencing was performed.
For direct cycle-sequencing, 45 µl of the PCR products were purified by the QIAquick PCR Purification Kit (Qiagen Ltd., Hilden, Germany) to obtain clean, double-stranded DNA amplificates. Sequencing reactions were performed by the BigDye Terminator Cycle Sequencing kit (Applied Biosystems) and analyzed on an ABI Prism 377 DNA sequencer (Applied Biosystems).
Purification of -Defensins
Synthetic HNP-1 peptide was purchased from Bachem Biochemica
(Heidelberg, Germany). Natural HNPs 1, -2, and -3 were isolated from 1
to 2 x 1010
peripheral blood mononuclear
cells (PBMCs) obtained by leukapheresis from healthy stem cell donors
who were pretreated with 10 µg/kg of granulocyte colony-stimulating
factor for 3 days. After MACS magnetic cell sorting (Miltenyi Biotec,
Bergisch-Gladbach, Germany) of CD34+
hematopoietic precursor cells, the residual leukocytes were used for
isolation of HNPs-1, -2, and -3 using the protocol previously described
with minor modifications.38
Briefly, around 1 x
1010
white blood cells of the leukapheresis
product were pelleted by centrifugation at 220 x g and
4°C for 10 minutes, washed once in PBS, and centrifuged. After
resuspension in 100 ml of PBS, erythrocytes were lysed by adding 300 ml
of EDTA-solution (4 mmol/L) and gently stirred for 1 minute. Lysis was
stopped by adding 100 ml of salt-solution [3.5% (w/v) NaCl, 4 mmol/L
EDTA]. After centrifugation for 20 minutes (220 x g),
the resulting cell pellet containing the leukocytes and blood cell
progenitors was resuspended in 20 ml of
Ca2+/EDTA-free Hank’s balanced salt solution
(with 2.5 mmol/L MgSO4 and 1 g glucose/L)
and homogenized in a Potter-Elvehjem homogenizer on ice (15 strokes,
600 rpm). After the addition of 100 ml of
Ca2+-free Hank’s balanced salt solution
containing 5 mmol/L EDTA, the cellular homogenate was centrifuged for
10 minutes at 1600 x g. Subsequently, the resulting
supernatant containing the leukocyte granules was ultracentrifuged at
27,000 x g for 20 minutes. The pellet was resuspended
in 5 to 10 ml of 5% (v/v) acetic acid and sonified eight times for 10
seconds on ice with a Sonifier B12 (Branson Ultrasonics Corp., Danbury,
CT) and diluted with 5% (v/v) acetic acid to a final volume of 100 ml.
This suspension was stirred for 12 hours on ice and ultracentrifuged at
27,000 x g for 20 minutes at 4°C. This extraction
step was repeated twice with 50 ml of 5% (v/v) acetic acid. The
defensin-containing supernatants were lyophilized and the resulting
crude product was purified by reversed phase-high performance liquid
chromatography (RP-HPLC) using a Nucleosil C18 column (150
x 4.6 mm, 3 µm; Grom, Herrenberg, Germany). The gradient was run
from 10 to 80% (v/v) acetonitrile/0.05% (v/v) trifluoroacetic acid
(TFA) with a flow-rate of 900 µl/min. The peak of the UV trace (214
nm) eluting at 
40% (v/v) acetonitrile was collected, lyophilized,
and shown by electrospray-ionization-mass spectrometry on a TSQ
700 (Finnigan, Bremen, Germany) to consist of a mixture of HNPs-1, -2,
-3 with a purity of more than 95%.
Detection of HNPs-1, -2, and -3 in a RCC Line
Cells (109) of the cultured RCC line TW33 were harvested and washed twice in PBS. The pelleted cells were resuspended in 10 ml of 10% (v/v) acetic acid, freeze-thawed on ice three times under vigorous shaking, and subsequently centrifuged at 1600 x g for 10 minutes at 4°C. The resulting supernatant was recentrifuged at 26,000 x g for 20 minutes at 4°C, lyophilized, and resolved in 0.05% TFA/water (v/v) and applied to RP-HPLC using a Grom-Sil 120 DDS4HE column (250 x 10 mm, 5 µm; Grom, Herrenberg, Germany). The gradient was run from 10 to 80% (v/v) acetonitrile/0.05% (v/v) TFA for 50 minutes with a flow rate of 2.2 ml/min, and the eluant was collected as 2-minute fractions. Four hundred µl of each fraction were tested for recognition by the HNP-1-, -2-, and -3-specific antibody DEF-3 in a slot-blot analysis. Briefly, the 400-µl fractions were drawn through a Nytran N transfer membrane (pore size 0.2 µm; Schleicher & Schuell, Dassel, Germany) in a Milli-Blot chamber (Millipore, Bedford, MA). The membrane was blocked for 1 hour with 2% powdered milk (m/v) in washing buffer [9% (m/v) NaCl, 10 mmol/L Tris-HCl, 0.05% (v/v) Tween 20, pH 7.8]. After washing the membrane was incubated with the DEF-3 mAb for 1 hour, washed again, and incubated for 1 hour with goat anti-mouse F(ab)2 fragment horseradish peroxidase (Dianova). The development was performed with the ECLplus Western blotting kit (Amersham Pharmacia Biotech). Positive fractions were concentrated in a SpeedVac and analyzed by MALDI mass spectrometry on a Voyager DE STR (Applied Biosystems, Langen, Germany).
Identification of HLA-DR-Associated Peptides
Preparation of HLA-associated peptide fractions was performed as
described previously.39
Briefly,
1010
interferon-
-treated RCC A-498 cells were
pelleted at 1000 x g and lysed with 2% (v/v) Triton
X-100. The lysate was cleared by ultracentrifugation and submitted to
affinity chromatography of HLA-DR molecules using the monoclonal
antibody L243. For prevention of nonspecific adsorbance, a mock
precolumn made of Sepharose material was used. Elution of the HLA
molecules and the bound peptides was performed using TFA/water, pH 2.0.
The peptide-released HLA-class II isolate was checked for purity by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Released
peptides were separated from the HLA molecules with an ultrafiltration
unit (Amicon, Danvers, MA) using a 20-kd cut-off membrane (Sartorius,
Göttingen, Germany). The peptide-containing fraction was
lyophilized and subsequently resolubilized in acetonitrile/water (1:1)
containing 0.1% (v/v) TFA.
Peptides were separated by microbore-reversed phase HPLC using a 125 x 2.1 mm C18 column (Vydac, Hesperia, CA) connected with an HPLC intelligent pump model L 6200 (Merck-Hitachi, Darmstadt, Germany) and a diode array detector model 1000S (Applied Biosystems). An elution gradient consisting of 0 to 80% acetonitrile/0.05% TFA for 70 minutes with a flow rate of 150 µl per minute was applied. From the absorbance trace at 220 nm, each fraction containing absorbance peaks of the eluate was collected and stored at -80°C until sequence analysis. All collected peaks have been examined for their peptide masses and for homogeneity by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). Pure fractions were submitted directly to an Edman sequencer. Sequencing results were compared with the Swissprot database at the European Bioinformatics Institute (Hinxton Hall, UK) (http://www.ebi.ac.uk/swissprot/).
Tumor Cell Proliferation Assays
RCCs (104/well) were incubated in triplicate in 96-well round-bottom microtiter plates (Greiner, Frickenhausen, Germany) with and without different concentrations of the synthesized HNP-1 or the isolated HNP-1, -2, and -3 peptide fraction (0 to 100 µg/ml) in RPMI 1640 (Life Technology, GmbH, Karlsruhe, Germany) supplemented with 2 mmol/L of L-glutamine, 25 mmol/L Hepes, 50 µg/ml gentamicin, without serum at 37°C and 5% CO2. After 24 hours cellular proliferation was quantified by measuring nuclear incorporation of tritiated thymidine ([3H] TdR) (specific activity 185 GBq/mmol used at 37 kBq/well; Amersham-Buchler, Braunschweig, Germany) after a 16-hour pulse label. Radioactivity was measured by liquid scintillation counting and data reduction using in-house programs. The results are expressed as mean corrected counts per minute (ccpm) and stimulation indices (SI; mean ccpm of the sample/mean ccpm of the control). Blocking studies of cell proliferation were performed by preincubation of defensin with the polyclonal or the monoclonal antibody DEF3.
Tumor cell proliferation was also assessed by evaluation of viable cells before and after incubation with HNPs-1, -2, and -3 using a colorimetric assay based on the cleavage of the tetrazolium salt WST-1 (Roche Molecular Biochemicals, Mannheim, Germany) by mitochondrial dehydrogenases. Tumor cells (104) cultured in 96-well flat-bottom microtiter plates under serum-free culture conditions with and without HNPs-1, -2, and -3 for 40 hours as described above were incubated with 20 µl of the ready-to-use WST-1 reagent in a final cell culture volume of 220 µl/well at 37°C and 5% CO2 for 10 minutes. After thoroughly shaking for 1 minute, formazan dye produced by metabolically active cells was determined by measuring the absorbance of the cell culture medium (RPMI 1640 without phenol red) against a background control (absorbance of culture medium plus WST-1 in the absence of cells) with an enzyme-linked immunosorbent assay reader at 440-nm wavelength. A calibration curve of formazan dye absorption was established with 3 x 104 to 8 x 102 titrated unmanipulated tumor cells. Cellular equivalents of the tumor cell test cultures were determined by comparison of their absorption values with the calibration curve.
Proliferation was also assessed by direct cell counting of tumor cells
cultured on 8-well Lab-Tek Permanox chamber slides (Nalge Nunc Int.
Corp., Naperville, IL) with and without 12.5 µg/ml of defensin in
duplicates in serum-free medium as described above. After 40 hours the
cell nuclei were stained on the slides with DAPI (1 µg/ml) and
evaluated by epifluorescence light microscopy (Olympus BX-60). The cell
nuclei of the tumor cells adherent to the bottom of each culture well
(0.25 cm2
per well) were quantified on digital
pictures taken from the blue fluorescence channel using the
Scananalytics Software IPLab Evaluation
(http://www.scananalytics.com).
Lymphocyte Proliferation Assay
An HLA-DRB1*0301-restricted T cell line isolated from a normal donor after priming of PBMCs with the Epstein-Barr virus-transformed HLA-DRB1*0301 homozygous B-cell line Cox was restimulated with untreated Cox and Cox pulsed with defensin. Cox cells (1 x 106) were preincubated with 35 µg/ml of HNPs-1, -2, and -3 peptide isolates in RPMI 1640 for 2 hours at 37°C. Pulsed Cox cells were washed twice with RPMI 1640 and irradiated with 80 Gy. Then 5 x 104 irradiated Cox cells with and without preincubation with defensin were co-cultured with 5 x 104 cells/well of the T cell line in round-bottomed microtiter plates for 1 or 3 days, respectively, at 37°C. After 24 hours, 48 hours, and 72 hours 37 kBq of [3H]-TdR (Amersham-Buchler, Braunschweig, Germany) was added and cells harvested after 16 hours on glass-fiber filtermats using a semiautomated cell harvester. T cell proliferation was determined by 3H-TdR incorporation by liquid scintillation counting. Identical plates were cultured and harvested at 24 hours, 48 hours, and 72 hours; all assays were performed in triplicate. The results are expressed as mean ccpm and SI (mean ccpm of the sample/mean ccpm of the control).
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Results |
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Five biopsies of kidneys from healthy donors were investigated by
indirect immunoperoxidase staining with two different murine mAbs as
well as with the polyclonal antibody specific for the -defensins
HNPs-1, -2, and -3. All tested antibodies against HNPs-1, -2, and -3
revealed two different staining patterns: patchy staining within
glomeruli as well as in the interstitium (Figure 1A)
and fine granular
intracytoplasmic labeling of proximal tubular epithelial cells (Figure 1C)
. The patchy staining correlated mostly with the presence of
granulocytes as demonstrated by labeling of consecutive sections with
antibodies against neutrophil elastase (Figure 1B)
and suggested that
HNPs-1, -2, and -3 were released into the surrounding tissues from the
neutrophils. Proximal epithelial cells stained for intracytoplasmic
granular deposits of -defensins often showed signs of cellular
necrosis. In control experiments, preincubation of the applied mAbs
with HNP-1, -2, and -3 isolates from leukapheresis products completely
blocked staining of normal renal tissue (data not shown). Triple
immunofluorescence labeling of normal kidney sections confirmed the
presence of HNPs-1, -2, and -3 in and at the surroundings of
elastase-positive neutrophils (Figure 2E)
, but also in proximal
tubular epithelial cells (Figure 2F)
. Occasionally, HNPs-1, -2, and -3
appeared to be also present in endothelial cells of some interstitial
capillaries and of small as well as larger arterial vessels.
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Thirty-one samples of different RCC tissues were investigated by
indirect immunoperoxidase labeling with both mAbs as well as the
polyclonal antibody specific for HNPs-1, -2, and -3. Two different
patterns of HNP-1, -2, and -3 presence in the analyzed tumors were
seen. In most of the analyzed RCC biopsies, random distribution of
patchy stains of HNP-1, -2, and -3 was noticed (Figure 1E)
. In those 25
tumors, the patchy stains for HNPs-1, -2, and -3 were mostly found to
be associated with the presence of PMNs located between tumor cells as
shown by staining for neutrophil elastase in consecutive sections
(Figure 1F)
. In six RCC samples, however, moderate to strong diffuse
labeling of all tumor cells was seen with the monoclonal and polyclonal
antibodies against HNPs-1, -2, and -3 (Figure 1G)
. Again, staining for
neutrophil elastase often showed neutrophils to be dispersed between
the malignant cells (Figure 1H)
, but adjacent to neutrophils, in
addition, epithelial tumor cells appeared to be positive for HNPs-1,
-2, and -3. In some parts of these tumors this strong labeling for
HNP-1, -2, and -3 deposits seemed to correlate with large areas of
cellular necrosis (Figure 1D)
. Because these tumors were diagnosed
grade II to III, diffuse expression of HNPs-1, -2, and -3 seemed to
correlate with a higher grade of tumor malignancy.
Triple-immunofluorescence labeling with antibodies against HNP-1, -2,
and -3, DAPI, and neutrophil elastase or cytokeratin 8 confirmed the
presence of defensins in association with neutrophils in most tumor
tissues (Figure 2G)
, but also showed that HNPs-1, -2, and -3 were
clearly present in single epithelial cells of 25 tumor
tissues (Figure 2A)
. Corresponding to the immunoperoxidase
staining a small number of RCC tissues showed the presence of HNPs-1,
-2, and -3 in almost all malignant cells also expressing cytokeratin 8
(Figure 2
; B, C, and D).
FACS Analysis of HNPs-1, -2, and -3 in RCC Lines
In FACS analyses, all eight RCC lines tested showed moderate to
strong intracellular expression of the -defensins after labeling
with the mAbs specific for HNPs-1, -2, and -3. Representative
histograms of intracellular labeling of HNPs-1, -2, and -3 are shown
for five RCC lines in comparison to the promyelocytic-positive control
cell line HL-60 (Figure 3)
.
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In RT-PCR analysis, the intron 2-spanning primers used allowed
amplification of specific HNPs-1, -2, and -3 mRNA products of 251 bp
from cDNA. The amplified fragments contained sequences coding for part
of the propiece (positions 112 to 242) as well as of the mature
peptides (positions 243 to 326) of HNPs-1, -2, and -3.37
HNP-1, -2, and -3 transcripts were constantly found in PBMCs of
leukapheresis products as well as in five of the eight tested RCC lines
(Figure 5)
. The transcripts were
confirmed by direct cycle sequencing to exactly match the expected
coding sequences of specific mRNA37
for HNP-1 or -3
products. However, the level of HNP-1, -2, and -3 transcripts varied
between the individual HNP-1, -2, and -3 mRNA-positive RCC lines as
well as between cultures of the same cell line from different time
periods. Freshly thawed cells usually showed stronger transcription of
the -defensin genes than long-standing cultures of these RCC lines.
The other three of the eight tested RCC lines A-498, 769-P, and LE 9211
as well as cultures of normal primary tubular cells TK163 revealed weak
expression of HNP-1, -2, and -3 mRNA in several experiments if freshly
thawed, but prolonged, cultures of these cells were negative. Presence
of HNP-1, -2, and -3 mRNA was also observed by RT-PCR analysis in five
RCC tissues as well as in two normal renal kidneys, but could be
expected to represent HNP-1, -2, and -3 mRNA from neutrophils also
(data not shown).
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Presence of HNP-1, -2, and -3 peptides in RCCs was
demonstrated by mass-spectrometric analysis of lysates of the RCC line
TW33. Mass-spectral profiling of acid-eluted proteins/peptides of the
lysate of this cell line by MALDI showed three prominent average mass
peaks for the mature peptides of HNP-1
(ACYCRIPACIAGERRYGTC-IYQGRLWAFCC) at m/z 3442.98 [theoretical average
mass (M + H)+3443.09u], HNP-2 differing from
HNP-1 only in the lack of alanine in the first position
(CYCRIPACIAGERRYGTCIYQGRLWAFCC) at m/z 3371.84 [theoretical average
mass (M + H)+3372.01u] and HNP-3 with an
aspartic acid instead of an alanine compared to HNP-1
(DCYCRIPACIAGERRYGTCIYQGRLWAFCC) at m/z 3486.93 [theoretical average
mass (M + H)+3487.10u] (Figure 6)
.
|
. The induced HLA-DR-peptide complexes were isolated from
the RCC line A-498 by immunoaffinity chromatography with the monoclonal
antibody L243. RP-HPLC analysis of the acid-eluted HLA-DR-bound
peptides revealed one dominant peptide fraction at the retention time
38.35 minutes that was shown by MALDI-MS analysis and consecutive Edman
sequencing to consist of the mature HNP-1 and -2 peptides (Figure 7)
.
|
To investigate a possible functional significance of -defensin
expression in RCC, different RCC lines were incubated in serum-free
RPMI 1640 medium with varying concentrations of the isolated natural
HNP-1, -2, and -3 peptide fraction. In addition, a synthetic peptide
HNP-1 was used. As shown in Figure 8A
,
incubation with the HNP-1, -2, and -3 peptide fraction at 6 to 25
µg/ml concentrations strongly stimulated DNA synthesis as
measured by 3H-thymidine incorporation after 24
hours in two (A-498 and 786-0) of five tested RCC lines in comparison
to untreated control cultures of the same cell lines. At higher
concentrations (>25 µg/ml), the mixture of -defensins revealed
suppression of DNA synthesis in all cell lines tested. Mean SIs of cell
proliferation in several repeated experiments were 4.9 and 1.9 for
A-498 and 786-0, respectively. Induction of cell proliferation (SI 3.2)
was also reproduced in the RCC line A-498 by incubation with 12.5
µg/ml of the synthetic peptide HNP-1. On the contrary, proliferation
of the RCC line A-498 induced by the -defensins could be inhibited
by preincubation of the HNP-1, -2, and -3 peptide fraction with the mAb
DEF3 (12.5 µg/ml) (Figure 8B)
.
|
, inset). On incubation with higher
concentrations of defensins (>25 µg/ml), all RCC lines showed a
strong decline of viable cells even below the levels of untreated
cultures. Direct counting of fluorescence-labeled cell nuclei of A498
tumor cells cultured on chamber slides also confirmed a significant
increase of total cell numbers after 40 hours of stimulation with 12.5
µg/ml of defensin in comparison to untreated tumor cell cultures
(Figure 8C)
. Modulation of Antigen-Presenting Function by HNPs-1, -2, and -3
Functional effects of -defensin binding to HLA-DR molecules on
immune recognition of renal carcinoma cells could not be directly
evaluated because the RCC lines were constitutively negative for
HLA-class II antigens. After stimulation with interferon- all RCC
lines except CRL 1932 expressed HLA-DR molecules, but were unable to
stimulate alloreactive proliferative T cell responses (data not shown).
Dominant endogenous binding of -defensins to HLA-DR molecules as
shown in the RCC line A-498, however, could not be blocked and
discriminated from other possible mechanisms inhibiting T cell
allorecognition of the tumor cells. Therefore, the Epstein-Barr virus
transformed B-cell line Cox was preincubated with 35 µg/ml of
defensins for 2 hours, washed, and used to stimulate an HLA-DR
restricted alloreactive T cell line in comparison to the unmanipulated
B cell line. After 24 hours, 48 hours, or 72 hours of co-culture of the
T cells and of the irradiated B cell line, 3H-TdR
was added and incorporated radioactivity was measured 16 hours later.
As shown in Figure 9
, the T cells were
highly reactive against the unmanipulated B cell line, but revealed a
13% reduced proliferative activity after 48 hours and 75% after 72
hours stimulation with Cox pulsed with defensins by comparison.
|
|
Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
-defensins HNP-1, -2, and -3 can represent peptide constituents of
renal carcinoma cells. By RT-PCR, immunohistological, and
mass-spectrometrical analysis HNPs-1, -2, and -3 were found to be
expressed in different RCC lines in culture. Of the six human
-defensins, HNPs-1, -2, and -3 have first been described to be
exclusively expressed in neutrophils and were only recently shown to be
also present in specific lymphocytes.15,20
In the kidney,
only ß-defensins have previously been detected in
tubuli.14,40
Through immunohistological analysis, HNPs-1,
-2, and -3 were shown to be also present in situ in normal
tubular epithelial cells of the kidney as well as in tumor cells of a
large group of RCC tissues adjacent to their expected expression in
neutrophils. With regard to their functional role in RCC, -defensins
were observed to possess mitogenic activity on a subset of RCC lines.
Identification of HNPs-1, -2, and -3 as peptides endogenously bound to
HLA-DR molecules on a RCC line suggested that -defensins may also
influence immune recognition of RCC.
By FACS analysis, all investigated RCC lines exhibited intracellular
labeling for -defensins at similar or lower levels as the myeloid
cell line HL-60. Presence of HNP-1, -2, and -3 in RCC lines was further
established by mass-spectrometric identification of the respective
mass-peaks for mature HNP-1, -2, and -3 peptides in cell lysates of the
RCC line TW33. Contrary to FACS analyses, only five RCC lines
repeatedly showed mRNA transcripts for HNP-1, -2, and -3 peptides
similar to HL-60 or PBMCs from leukapheresis products. However, the
other three RCC lines tested varied in their positivity for HNP-1, -2,
and -3 mRNA in different subcultures. Although the -defensin HNPs-1,
-2, and -3 have been claimed to represent microbicidal peptides with
exclusive cell lineage specificity for hematopoetic cells such as
neutrophils and some lymphocytes, specific genomic elements or
transcription factors controlling myeloid lineage-restricted gene
activation of -defensins have not yet been
identified.41,42
Our observation that the amount of
-defensin mRNA varied greatly between different RCC lines and was
not stable in the same RCC line if tested after different culture
periods may reflect a complex, strictly controlled regulation of HNP-1,
-2, and -3 mRNA, as it has been also claimed for myeloid
cells.42
Although -defensin transcription may be
modulated by certain cytokines such as IL-8 (Mueller CA, Flad T, Klatt
T, Steiert I, Beir H, Mueller GA, manuscript in preparation),
regulatory mechanisms responsible for the observed temporal or
individual variations of -defensin mRNA expression in RCC lines are
still unknown. In this respect, it may also be of interest that a loss
of heterozygosity at chromosome 8p23, which may include the defensin
gene cluster,43
in RCC tumors could play a role in the
regulation of -defensin expression and tumor
progression.44
By triple immunohistological stainings of biopsies from normal kidneys
and RCC tissues, HNPs-1, -2, and -3 were found to be also present
in situ in normal as well as malignant renal epithelial
cells co-expressing cytokeratin. In the normal kidney, presence of
HNP-1, -2, and -3 peptides appeared to be concentrated to proximal
tubular cells in contrast to reports on ß-defensins primarily
localized to epithelial layers of the loops of Henle, the distal
tubuli, and the collecting duct.14
Although weak
expression of HNP-1-, -2-, and -3-specific mRNA had been observed in
subcultures of primary tubular epithelial cells, we could not entirely
exclude that the labeling of proximal tubular cells in the normal
kidney also reflected reabsorption of -defensins from the primary
glomerular ultrafiltrate. As with ß-defensins, HNPs-1, -2, and -3
might contribute to the local antimicrobial defense of the nephron and
thus participate in protection from ascending urinary infections in the
normal kidney. HNPs-1, -2, and -3, however, were not only found in
tubular epithelial cells of the normal kidney, but also in neutrophils
double-immunostained for neutrophil elastase as expected. PMNs were
rarely present within the tubulointerstitial space and also within the
glomerular capillary loops. Both mAbs specific for HNPs-1, -2, and -3
labeled intracellular granules but, surprisingly, also halos
surrounding such neutrophils in the normal renal tissues. Such patchy
stains suggested release of HNPs-1, -2, and -3 from neutrophils and
diffusion into the encircling parenchyma, as it has been reported for
brain tissue.45
In 25 of 31 RCC tissues tested a similar patchy pattern of HNP-1, -2,
and -3 immunostaining was observed. The -defensin patches mostly
correlated with the presence of single elastase-positive
polymorphonuclear cells in their center and several tumor epithelial
cells clearly double-stained for cytokeratin in their surroundings.
Unfortunately in all cases, we could not discriminate in
situ, whether HNPs 1, -2, and -3 were sequestered from adjacent
located neutrophils or also produced by the tumor cells. However,
because of the in vitro finding of defensin synthesis in the
RCC lines, and in particular in the RCC line TW33 that was established
from an analyzed tumor biopsy, malignant epithelial cells contributing
to HNP-1, -2, and -3 synthesis is most likely.
-Defensin-synthesizing tumor cells appeared to be primarily present
in six RCC biopsies of different histological subtypes in which all
epithelial tumor cells were stained diffusely and strongly for HNPs-1,
-2, and -3.
Interestingly, cytolytic effects in tissues were occasionally
correlated with presence of the -defensins. In particular, groups of
tumor cells in the center of primarily extended defensin patches tended
to exhibit morphological signs of necrosis. Although the in
vivo implications of defensin expression in RCC are still unknown,
in vitro experiments on RCC lines suggested that, depending
on the amount of HNP-1, -2, and -3 present in the tumor tissue, HNPs-1,
-2, and -3 may exert potent mitogenic activity on tumor cells or elicit
cytotoxic effects. At higher concentrations than 25 µg/ml, HNPs-1,
-2, and -3 exerted cytotoxic effects on all tested RCC lines in an
in vitro serum-free culture system, but interestingly at
lower concentrations they stimulated cell growth of a subset of tumor
cell lines. Similar mitogenic effects of defensins have been previously
described on mouse fibroblasts and epithelial cells.25
According to our in vitro and in vivo
observations, RCCs may be heterogeneous in their content of tumor cells
that are sensitive or resistant to stimulation with -defensins.
Thus, it is indeed plausible that -defensins could contribute to
tumor cell selection by promoting proliferation of a subset of
malignant cells. At present it is unknown whether such selection
mechanisms could favor RCC subpopulations that produce -defensins
and are associated with a higher grade of malignancy.
In neutrophils -defensins are initially synthesized as inactive
prodefensins with an NH2-terminal neutralizing
propiece that is cleaved by as yet unknown proteases. From our
immunocytological studies with antibodies recognizing the mature as
well as the immature peptides we could not determine whether renal
carcinoma cells in tissues contain prodefensins or the mature HNP-1,
-2, and -3 peptides. At least from mRNA analysis prodefensins were most
likely to be synthesized in RCC lines and mass-spectrometric analysis
had shown that also low amounts of the mature HNP-1, -2, and -3
peptides were also present. From an analysis of RCC lines by confocal
microscopy, we recognized that -defensins were not confined to a
specific subcellular compartment in RCC lines as in contrast to
neutrophils. This finding suggested that RCC lines might accumulate
-defensins mainly as precursor peptides to avoid self-destruction.
It is, however, still unknown how RCC lines synthesizing -defensins
are able to process these peptides. As with the defensins HD5 and HD6
in intestinal epithelial cells,46
processing of HNP-1, -2,
and -3 precursors could rely on the concomitant expression of specific
proteases such as the metalloprotease matrilysin. Further analyses are
required to elucidate such regulatory mechanisms controlling the level
of functional defensin production in RCC.
As another consequence of HNP-1, -2, and -3 production, endogenous
binding of these peptides to HLA-DR molecules in RCC lines could be
found after stimulation of the malignant cells with interferon-. As
it has been previously shown, -defensins were observed to
down-regulate HLA-class II-specific alloreactive T cell responses after
preincubation of the stimulator cells with the HNP-1, -2, and -3
isolate in a dose-dependent manner.28
Also in this study,
proliferative responses of an alloreactive T cell line were reduced
after pulsing the stimulating cells with defensins. This indicated that
defensins bound to HLA-class II molecules in RCC as in other tumor cell
lines could inhibit major histocompatibility complex class
II-restricted RCC recognition by T cells. This could also account for
the known difficulties in using RCC lines as stimulatory cells of
tumor-reactive T cells in vitro.
Although it is claimed that defensins have their predominant functional
role as neutrophil-specific endogenous antibiotics in
humans,3
several other activities, in particular
chemotactic properties for immune cells, have been attributed to these
peptides.22,23
This study provides evidence for the first
time that -defensins may also be expressed in specific epithelial
cells of the kidney and possibly involved in the pathogenesis of renal
tumors. By influencing tumor cell proliferation and immune recognition,
-defensins could potentially modulate tumor progression of RCC.
|
Acknowledgements |
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Footnotes |
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Supported by the Dr. Mildred Scheel Foundation, Germany (grant 10-1561).
Accepted for publication January 2, 2002.
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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defensins are expressed by specific lymphocyte and monocyte populations. Blood 2000, 96:3086-3093-defensin activation by the metalloproteinase matrilysin in innate host defense. Science 1999, 286:113-117This article has been cited by other articles:
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  N. Xu, Y.-s. Wang, W.-b. Pan, B. Xiao, Y.-j. Wen, X.-c. Chen, L.-j. Chen, H.-x. Deng, J. You, B. Kan, et al. Human {alpha}-defensin-1 inhibits growth of human lung adenocarcinoma xenograft in nude mice Mol. Cancer Ther., June 1, 2008; 7(6): 1588 - 1597. [Abstract] [Full Text] [PDF]
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 P. Hubert, L. Herman, C. Maillard, J.-H. Caberg, A. Nikkels, G. Pierard, J.-M. Foidart, A. Noel, J. Boniver, and P. Delvenne Defensins induce the recruitment of dendritic cells in cervical human papillomavirus-associated (pre)neoplastic lesions formed in vitro and transplanted in vivo FASEB J, September 1, 2007; 21(11): 2765 - 2775. [Abstract] [Full Text] [PDF]
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 J. Markovic-Lipkovski, C. A. Muller, G. Klein, T. Flad, T. Klatt, S. Blaschke, J. T. Wessels, and G. A. Muller Neural cell adhesion molecule expression on renal interstitial cells Nephrol. Dial. Transplant., June 1, 2007; 22(6): 1558 - 1566. [Abstract] [Full Text] [PDF]
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 M. J. Nam, M. K. Kee, R. Kuick, and S. M. Hanash Identification of Defensin {alpha}6 as a Potential Biomarker in Colon Adenocarcinoma J. Biol. Chem., March 4, 2005; 280(9): 8260 - 8265. [Abstract] [Full Text] [PDF]
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 J. Aarbiou, R. M. Verhoosel, S. van Wetering, W. I. de Boer, J. H. J. M. van Krieken, S. V. Litvinov, K. F. Rabe, and P. S. Hiemstra Neutrophil Defensins Enhance Lung Epithelial Wound Closure and Mucin Gene Expression In Vitro Am. J. Respir. Cell Mol. Biol., February 1, 2004; 30(2): 193 - 201. [Abstract] [Full Text] [PDF]
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