An Antimicrobial Peptide Regulates Tumor-Associated Macrophage Trafficking via the Chemokine Receptor CCR2, a Model for Tumorigenesis

Abstract

Background
Tumor-associated macrophages (TAMs) constitute a significant part of infiltrating inflammatory cells that are frequently correlated with progression and poor prognosis of a variety of cancers. Tumor cell-produced human β-defensin-3 (hBD-3) has been associated with TAM trafficking in oral cancer; however, its involvement in tumor-related inflammatory processes remains largely unknown.

Methodology
The relationship between hBD-3, monocyte chemoattractant protein-1 (MCP-1), TAMs, and CCR2 was examined using immunofluorescence microscopy in normal and oral carcinoma in situ biopsy specimens. The ability of hBD-3 to chemoattract host macrophages in vivo using a nude mouse model and analysis of hBD-3 on monocytic cell migration in vitro, applying a cross-desensitization strategy of CCR2 and its pharmacological inhibitor (RS102895), respectively, was also carried out.

Conclusions/Findings
MCP-1, the most frequently expressed tumor cell-associated chemokine, was not produced by tumor cells nor correlated with the recruitment of macrophages in oral carcinoma in situ lesions. However, hBD-3 was associated with macrophage recruitment in these lesions and hBD-3-expressing tumorigenic cells induced massive tumor infiltration of host macrophages in nude mice. HBD-3 stimulated the expression of tumor-promoting cytokines, including interleukin-1α (IL-1α), IL-6, IL-8, CCL18, and tumor necrosis factor-α (TNF-α) in macrophages derived from human peripheral blood monocytes. Monocytic cell migration in response to hBD-3 was inhibited by cross-desensitization with MCP-1 and the specific CCR2 inhibitor, RS102895, suggesting that CCR2 mediates monocyte/macrophage migration in response to hBD-3. Collectively, these results indicate that hBD-3 utilizes CCR2 to regulate monocyte/macrophage trafficking and may act as a tumor cell-produced chemoattractant to recruit TAMs. This novel mechanism is the first evidence of an hBD molecule orchestrating an in vivo outcome and demonstrates the importance of the innate immune system in the development of tumors.

Full text

An Antimicrobial Peptide Regulates Tumor-Associated
Macrophage Trafficking via the Chemokine Receptor
CCR2, a Model for Tumorigenesis
Ge Jin
1,5
*, Hameem I. Kawsar
1
, Stanley A. Hirsch
2
, Chun Zeng
3
, Xun Jia
1
, Zhimin Feng
1
, Santosh K.
Ghosh
1
, Qing Yin Zheng
4,5
, Aimin Zhou
3
, Thomas M. McIntyre
6
, Aaron Weinberg
1
1Department of Biological Sciences, Case Western Reserve University School of Dental Medicine, Cleveland, Ohio, United States of America, 2Department of Oral
Pathology, Case Western Reserve University School of Dental Medicine, Cleveland, Ohio, United States of America, 3Department of Chemistry, Cleveland State University,
Cleveland, Ohio, United States of America, 4Department of Otolaryngology-Head and Neck Surgery, University Hospitals of Cleveland, Case Western Reserve University
School of Medicine, Cleveland, Ohio, United States of America, 5Case Comprehensive Cancer Center, Cleveland, Ohio, United States of America, 6Department of Cell
Biology, Lerner Research Institute, Cleveland Clinic College of Medicine of Case Western Reserve University, Cleveland, Ohio, United States of America
Abstract
Background:
Tumor-associated macrophages (TAMs) constitute a significant part of infiltrating inflammatory cells that are
frequently correlated with progression and poor prognosis of a variety of cancers. Tumor cell-produced human b-defensin-3
(hBD-3) has been associated with TAM trafficking in oral cancer; however, its involvement in tumor-related inflammatory
processes remains largely unknown.
Methodology:
The relationship between hBD-3, monocyte chemoattractant protein-1 (MCP-1), TAMs, and CCR2 was
examined using immunofluorescence microscopy in normal and oral carcinoma in situ biopsy specimens. The ability of hBD-
3 to chemoattract host macrophages in vivo using a nude mouse model and analysis of hBD-3 on monocytic cell migration
in vitro, applying a cross-desensitization strategy of CCR2 and its pharmacological inhibitor (RS102895), respectively, was
also carried out.
Conclusions/Findings:
MCP-1, the most frequently expressed tumor cell-associated chemokine, was not produced by tumor
cells nor correlated with the recruitment of macrophages in oral carcinoma in situ lesions. However, hBD-3 was associated
with macrophage recruitment in these lesions and hBD-3-expressing tumorigenic cells induced massive tumor infiltration of
host macrophages in nude mice. HBD-3 stimulated the expression of tumor-promoting cytokines, including interleukin-1a
(IL-1a), IL-6, IL-8, CCL18, and tumor necrosis factor-a(TNF-a) in macrophages derived from human peripheral blood
monocytes. Monocytic cell migration in response to hBD-3 was inhibited by cross-desensitization with MCP-1 and the
specific CCR2 inhibitor, RS102895, suggesting that CCR2 mediates monocyte/macrophage migration in response to hBD-3.
Collectively, these results indicate that hBD-3 utilizes CCR2 to regulate monocyte/macrophage trafficking and may act as a
tumor cell-produced chemoattractant to recruit TAMs. This novel mechanism is the first evidence of an hBD molecule
orchestrating an in vivo outcome and demonstrates the importance of the innate immune system in the development of
tumors.
Citation: Jin G, Kawsar HI, Hirsch SA, Zeng C, Jia X, et al. (2010) An Antimicrobial Peptide Regulates Tumor-Associated Macrophage Trafficking via the Chemokine
Receptor CCR2, a Model for Tumorigenesis. PLoS ONE 5(6): e10993. doi:10.1371/journal.pone.0010993
Editor: Jo
¨rg Hermann Fritz, University of Toronto, Canada
Received December 8, 2009; Accepted May 17, 2010; Published June 8, 2010
Copyright: ß2010 Jin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a Scientist Development Grant #0535088N from the American Heart Association (http:www.americanheart.org) (GJ) and
Grant #IRG-91-022-15 from the American Cancer Society (wwww.cancer.org) (GJ), NIH/NIDCR P01DE019089 and P01DE019759 (AW), and NIH/R01DC007392 and
NIH/NIDCD R01DC009246 (QZ) from the National Institutes of Health (www.nih.gov). The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: ge.jin@case.edu
Introduction
Collaborative interactions of tumor cells with leukocyte
infiltrates in the tumor microenvironment can significantly
influence tumor development and progression [1,2,3,4]. Macro-
phages residing in the tumor site, collectively termed tumor-
associated macrophages (TAMs), often constitute a major part of
infiltrating leukocytes and represent a significant component of
cancer-associated inflammatory environment [5]. Clinical studies
have shown that tumor infiltration of macrophages is associated
with progression and poor prognosis in more than 80% of cancers,
including cancers of breast, prostate, bladder, cervix, and head
and neck [6,7]. Experimental studies using mouse models confirm
that genetic and chemical ablation of macrophages leads to an
inhibition of tumor progression and reduced rate of metastasis
[8,9,10,11]. Tumor-produced factors, including a variety of
cytokines, activate TAMs to stimulate tumor cell proliferation,
migration, angiogenesis, metastasis [5,8,12]. TAMs derive from
circulating monocytes that are selectively recruited to the tumor
site by chemotactic factors locally produced by tumor and stromal
PLoS ONE | www.plosone.org 1 June 2010 | Volume 5 | Issue 6 | e10993

cells. Experimental and clinical studies have shown that monocyte
chemoattractant peptide-1 (MCP-1), also known as chemokine (C-
C motif) ligand 2 (CCL2), is perhaps the chemokine most
frequently expressed by tumor cells and is correlated with
recruitment of host macrophages to the tumor site in a variety
of human tumors, such as sarcomas, gliomas, melanomas, cancers
of the breast, cervix, and ovary [6,7]. Other chemokines, such as
CCL5, CCL7, CCL8, and CXCL12, as well as tumor cell-
produced growth factors, such as vascular endothelial growth
factor (VEGF), transforming growth factor-b(TGF-b), and
fibroblast growth factor (FGF), are also described as chemotactic
for monocytes/macrophages during tumor development [7,13].
These chemotactic factors, based on clinical and experimental
studies, are summarized in Table 1. Although lipopolysaccharides
(LPS) treatment induces production of MCP-1 and CCL20, as well
as IL-6 in cultured oral cancer cell lines in vitro, the contribution of
these cytokines in recruiting monocyte-lineage cells in oral
carcinoma in situ and invasive OSCC in vivo is still unknown
[14]. In head and neck squamous cell carcinoma (HNSCC),
infiltration of macrophages into and around cancer tissues is
significantly correlated with tumor size, aggressiveness, invasion,
and poor prognosis [15,16,17]. However, chemotactic molecules
that participate in the recruitment of inflammatory cells in
HNSCC are still largely undetermined. In oral squamous cell
carcinoma (OSCC), the expression of MCP-1 and CCL5 have
detected in scattered non-neoplastic inflammatory cells, while only
a few of MCP-1 expressing tumor cells have been found in less
than 40% of cases studied [18]. Therefore, the collective findings
to date suggest that other tumor cell-produced factors may
chemoattract immune cells. Our recent findings have shown that
tumor cells overwhelmingly produce human b-defensin-3 (hBD-3),
but not MCP-1, in the oral CIS lesion [19]. Kesting et al. have
confirmed our observations by reporting overexpression of hBD-3
in oral cancer tissues using paired cancerous and noncancerous
specimens derived from 46 patients [20]. HBD-3, therefore, may
play an important role in the development and progression of oral
cancer.
Human b-defensins (hBDs) are small cationic peptides originally
identified from the plasma of patients with renal disease (hBD-1)
and from psoriatic skin lesions (hBD-2 and hBD-3) as antimicro-
bial agents of innate immunity [21,22,23]. It has now been
reported that hBDs display a variety of biological activities,
particularly their participation in ‘‘cross-talking’’ with the adaptive
immune system [24]. HBD-3 has been shown to antagonize the
HIV co-receptor CXC chemokine receptor 4 (CXCR4) and to
activate professional antigen-presenting cells (APCs) via hetero-
dimerized TLR1 and 2 [25,26]. The chemokine receptor CCR6
has been shown to mediate migration of memory T cells and iDCs
in response to hBD-1 and -2 [27]. However, the membrane
receptor(s) that mediates hBD-3-induced monocytic cell migration
has yet to be identified, as CCR6 is not expressed on this cell type
[19,28,29]. Although the in vitro information being gathered to
date implies that hBDs have the capacity to immunoregulate the
adaptive immune system, whether they participate in actual
regulation of immune responses in vivo; i.e., inflammatory processes
in tumor pathogenesis, is largely unknown. We previously
demonstrated that hBD-3 over-expression by tumor cells in oral
CIS lesions is correlated with recruitment and infiltration of
macrophages to the tumor site [19]. In addition, hBD-3
chemoattracts monocytic THP-1 cells in vitro, suggesting a possible
connection between hBD-3 expression and TAM trafficking [19].
In the present study, we demonstrate that tumor cells do not
express MCP-1 in oral CIS biopsies. Moreover, xenograft tumors
generated by tumorigenic cells that overexpress hBD-3 show
massive host macrophage infiltration and enhanced tumorigenicity
when compared with those formed by parent cells. In addition,
hBD-3 induced monocytic cell migration is blocked by cross-
desensitization with MCP-1 and by the treatment of cells with the
specific CCR2 inhibitor, RS102895, respectively. Collectively,
these results support our hypothesis that hBD-3 functions as a
chemoattractant to recruit macrophages and that CCR2 plays a
central role in mediating monocyte/macrophage migration in
response to hBD-3.
Results
Association of the hBD-3-rich tumor environment with
intratumoral accumulation of CCR2 expressing cells
We previously demonstrated that tumor cells overwhelmingly
express hBD-3, but not hBD-2, in the oral CIS lesion [19]. To
confirm the observation, additional normal (Figure 1A; one
representative sample is shown in hematoxylin and eosin [H&E]
Table 1. Chemotactic molecules involved in inflammatory cell trafficking.
Chemoattractants Receptors Associated tumors References
MCP-1/CCL2 CCR2 sarcoma, gliomas, melanomas, lung, breast, cervix, ovary, colon. [6,7,72,73]
CCL3 CCR5 lung, breast, hepatocellular carcinoma, multiple myeloma,
chronic lymphocytic leukemia, colon.
[73,74,75,76,77]
CCL4 CCR5 chronic lymphocytic leukemia, colon. [75,76]
CCL5/RANTES CCR1, CCR5 breast, melanoma, cervix [78,79,80,81,82]
CXCL8/IL-8 IL8R melanoma, lung [72]
CXCL12/SDF1-aCXCR4 breast, ovarian [83,84]
M-CSF CSF1R sarcoma [85,86]
GM-CSF CSF2R breast and others [87,88,89]
VEGFA VEGFR1 lung and others [90,91,92]
TGFbTGFR breast, lung [91,93,94]
C5a C5AR1 cervix [95,96,97]
RANTES, Regulated upon Activation Normal T-cell Expression and presumably Secreted; C5a, complement 5a; M-CSF, Macrophage Colony-Stimulating Factor; GM-CSF,
Granulocyte-Macrophage Colony Stimulating Factor.
doi:10.1371/journal.pone.0010993.t001
hBD-3 Regulates TAM via CCR2
PLoS ONE | www.plosone.org 2 June 2010 | Volume 5 | Issue 6 | e10993

staining) and CIS biopsies (Figure 1B; one representative is shown
in H&E staining) were used for co-immunofluorescence staining
using antibodies to hBD-2 and hBD-3. In normal oral epithelia,
hBD-3 expression was detected only in the basal layers, while
hBD-2 production could be observed in the differentiated
superficial layers (Figure 1C; four samples are shown). However,
in CIS, hBD-3 expression was observed throughout the lesion site
in all biopsy samples, while hBD-2 expression was absent
(Figure 1D; seven samples are shown; compare Figure 1C with
Figure 1D). Immunofluorescence staining with the isotype control
antibody to hBD-2 or hBD-3 did not detect any signals
(Figure 1F). To compare the expression of hBDs between normal
oral epithelia and CIS lesions quantitatively, immunofluorescence
intensities of hBD-2 and hBD-3 were measured and normalized
with that of nuclei in the epithelia of CIS and normal samples.
The expression of hBD-3 was ,3.7-fold higher in CIS samples
(n= 7) compared with that in normal epithelia (n= 4), whi le hBD-
2 expression was significantly higher in normal oral epithelia than
CIS biopsies (Figure 1E). These results, in conjunction with those
of Kesting et al [20], further support our original observation of
the presence of an hBD-3-rich tumor microenvironment in CIS
lesions.
Figure 1. Expression of hBD-2 and hBD-3 in normal and carcinoma
in situ
(CIS) epithelia. (A and B) H&E images in normal oral epithelium
(A) and CIS lesion (B) biopsy samples. (C and D) Immunofluorescent images of hBD-2 (green) and hBD-3 (red) in normal (C) and CIS (D) oral epithelial
biopsies. Nuclei, blue (DAPI). (E) Quantification of immunofluorescence intensities of hBD-2 and hBD-3 over that of nuclei in normal oral epithelia and
CIS biopsies. Epithelial biopsies were derived from 4 normal (n= 4) and 7 CIS (n= 7) individuals. The line drawn through the boxplot graph (E)
represents the mean of the results and the line extending vertically from the box indicates the lowest and highest value in the data set. *, p= 0.00,
** p= 0.00. In normal oral epithelia, the mean of hBD-2 and hBD-3 was 1.53 and 2.11, respectively. In CIS tissues, however, the mean of hBD-2 and
hBD-3 was 0.70 and 5.63, respectively. (F) Isotype controls for hBD-2 (left) and hBD-3 (right) using anti-goat IgG and anti-rabbit IgG antibodies,
respectively. Nuclei, blue (DAPI).
doi:10.1371/journal.pone.0010993.g001
hBD-3 Regulates TAM via CCR2
PLoS ONE | www.plosone.org 3 June 2010 | Volume 5 | Issue 6 | e10993

We have shown the association of hBD-3 with recruitment and
infiltration of macrophages, but not CD3+lymphocytes, into the
tumor site [19]. Since macrophages express the chemokine
receptor CCR2 for migration, we decided to determine whether
hBD-3 expression was associated with recruitment of CD68+/
CCR2+cells. Figure 2 shows the expression of hBD-3 in the CIS
lesion (Figure 2A and 2B) and co-stained CD68 and CCR2
accumulating in the CIS site, but not in the apparently normal
region adjacent to the lesion (Figure 2C; the enlarged inset on the
left, normal region adjacent to the CIS; on the right, CIS lesion,
white arrowheads indicate CCF2+macrophages), suggesting the
association of the hBD-3-rich tumor microenvironment with
recruitment of CCR2+macrophages.
Dissociation of expression of MCP-1 and tumor
recruitment of macrophages
Since MCP-1 has been shown to be produced by tumor cells
and correlated with recruitment and infiltration of immune cells in
a variety of cancers [30], we investigated expression of MCP-1 and
macrophage infiltration, using the macrophage cell surface marker
CD68, to determine if this linkage is upheld in oral CIS. MCP-1
was either undetectable or sporadically expressed in the epithelium
and lamina propria of normal oral tissue biopsy samples
(Figure 3A) and no macrophages were detected (Figure 3A; two
samples are shown). In clinically diagnosed CIS samples, however,
macrophages were recruited abundantly to and infiltrated into the
lesion site (Figure 3B and 3C). Interestingly, patterns of MCP-1
expression in these CIS samples were not consistent, nor
correlated with recruitment and infiltration of macrophages
(Figure 3B–D). In the CIS sample shown in Figure 3B, MCP-1
producing cells were located in the lamina propria adjacent to the
basement membrane of the CIS lesion, while recruited macro-
phages were present in the area and infiltrated into the lesion site,
where MCP-1 was not expressed (Figure 3B, enlarged inset; white
arrows above the lamina propria). The CIS biopsy from a different
patient showed no MCP-1 expression either in the lamina propria
or in the CIS site, while intratumoral macrophages were evident
(Figure 3C, enlarged inset). In another CIS sample, however,
MCP-1 expression was observed in the normal region adjacent to
the CIS, but not in the lesion (Figure 3D; enlarged inset on the left,
normal region adjacent to the CIS lesion, MCP-1 expression is
indicated with white arrows; enlarged inset on the right, the CIS
site). Staining of the consecutive section shown in Figure 3D with
antibodies to CCR2 and CD68 indicated the accumulation of
CCR2+macrophages in the CIS lesion (Figure 2C, enlarged inset
on the right). Notably, in all seven CIS samples tested, tumor cells
did not produce MCP-1 (Figure 3B–3D, enlarged insets). These
results suggest that the expression of MCP-1 in CIS lesions varies
and that there is no apparent correlation between MCP-1
expression and recruitment and infiltration of macrophages in
oral tumors.
Association of hBD-3 with tumorigenicity and tumor
infiltration of host macrophages in nude mice
To examine the in vivo role of hBD-3 in macrophage trafficking,
we inoculated nude mice subcutaneously with tumorigenic human
embryonic kidney 293 cells (HEK293) with or without hBD-3
overexpression. The transcription of hBD-3 in parent and hBD-3
overexpressing HEK293 cells was determined by RT-PCR, while
secreted and cell-associated hBD-3 peptide in these cells were
measured by ELISA analysis. The expression of hBD-3 mRNA
was undetectable in parent HEK293 cells, while clearly detectable
Figure 2. Localization of CCR2
+
/CD68
+
macrophages in the CIS lesion. (A) H&E image of a CIS biopsy specimen. The CIS lesion and the
adjacent normal region are demarcated. (B) Immunofluorescent staining of hBD-3 (red) in the consecutive section derived from (A). Dashed yellow
line, boundary separating the CIS and adjacent normal region; nuclei, blue (DAPI). (C) Co-immunofluorescent image of CCR2 (red) and CD68 (green) in
a consecutive section of (B). Several CCR2+/CD68 positive cells are indicated by white arrowheads (enlarged inset on the right) in the CIS lesion site.
Dashed yellow line, boundary separating the CIS and adjacent normal region; dashed white line, basement membrane; nuclei, blue (DAPI). (D) Isotype
controls of CCR2 (left panel) and CD68 (right panel) using sections derived from the same block of (B). Nuclei, blue (DAPI).
doi:10.1371/journal.pone.0010993.g002
hBD-3 Regulates TAM via CCR2
PLoS ONE | www.plosone.org 4 June 2010 | Volume 5 | Issue 6 | e10993

Figure 3. Localization of macrophages and MCP-1 expression in normal and CIS biopsy specimens. (A) Double-immunofluorescence
of CD68 (green) and MCP-1 (red) in two normal oral epithelial biopsies. Nuclei, blue (DAPI). (B) CD68 (green) and MCP-1 (red) in a CIS biopsy
section. Arrows, macrophages; arrowheads, MCP-1 expressing cells; dashed white line, basement membrane; nuclei, blue (DAPI). (C) CD68 (green)
and MCP-1 (red) in a CIS biopsy section derived from a second patient. MCP-1 is undetectable in the entire section. Arrows in enlarged inset,
macrophages; dashed white line, the basement membrane; nuclei, blue (DAPI). (D) Immunofluorescence of MCP-1 (red) in the CIS section (obtained
from the same block in Figure 2B) derived from a third patient. MCP-1 expressing cells (enlarged inset on the left, white arrows) are detected in the
normal region adjacent to the CIS site, but not in the CIS lesion. Dashed white line, basement membrane; dashed yellow line, boundary separating
the CIS (CIS) and adjacent normal region (N); nuclei, blue (DAPI). (E) Isotype control for MCP-1 (upper panel) and CD68 (lower panel), respectively.
Nuclei, blue (DAPI).
doi:10.1371/journal.pone.0010993.g003
hBD-3 Regulates TAM via CCR2
PLoS ONE | www.plosone.org 5 June 2010 | Volume 5 | Issue 6 | e10993

in the engineered hBD-3 overexpressing cell line (Figure 4A). The
production of hBD-3 peptide in culture medium and the cell lysate
was significantly higher in HEK293 cells that overexpressed hBD-
3 compared with parent HEK293 cells (Figure 4B and Figure 4C),
indicating that parent HEK293 cells do not express hBD-3. Ten
days after inoculation, xenograft tumors formed in mice inoculated
with parent HEK293 or hBD-3 overexpressing cells. However,
hBD-3 overexpressing cells generated larger tumors than those
established by parent cells (Figure 4D and 4E). In addition, hBD-3
overexpressing cells formed tumors in all eight inoculated sites,
while only four tumors were established in nude mice injected with
parent HEK293 cells (Figure 4F). The mean volumes for hBD-3
overexpressing tumors were about 66.9 mm
3
, while the mean
parent HEK293 tumor volume was 27.9 mm
3
(Figure 4F). These
results suggest that hBD-3 overexpression increases the incidence
of xenograft tumor formation and rate of tumor growth in nude
mice.
H&E staining of the formalin-fixed, paraffin-embedded sections
of the xenografts revealed a lighter-staining region within the
central area of the hBD-3 overexpressing tumor (Figure 5A, right
panel). However, this was not observed in tumors established by
parent HEK293 cells (Figure 5A, left panel). Histological analysis
indicated that the hBD-3 overexpressing xenograft tumors
featured non-encapsulated circumscribed tumor nodules that
contained relatively large, ovoid nuclei, mitotic structures, and
possible necrotic regions (Figure 5B). The lighter-staining region,
therefore, suggests necrosis in hBD-3 overexpressing tumors. To
determine whether hBD-3 chemoattracts host macrophages,
xenograft tumor sections were stained with the monoclonal
antibody to F4/80 antigen, a murine specific macrophage marker
[31]. The results showed massive host macrophage infiltration in
tumors generated with hBD-3 overexpressing cells, but not in
tumors formed by parent cells (Figure 5C, enlarged inset).
Interestingly, in hBD-3 overexpressing tumors, macrophages were
recruited preferentially to the area where the necrotic features
were evident, suggesting a correlation between macrophages
trafficking and necrosis (compare Figure 5A, right panel and
Figure 5C, upper right panel). Double-staining of the specimen
with antibodies to F4/80 and mouse CCR2 indicated infiltration
of CCR2+mouse macrophages in the tumor established by hBD-3
overexpressing cells (Figure 5D, CCR2+mouse macrophages are
indicated with white arrows). However, these cells were absent in
the tumor formed by parent HEK293 cells (Figure 5E). Clearly,
hBD-3 was expressed at high levels in tumors established from the
hBD-3 overexpressing cells (Figure 5F).
Induction of cytokine expression by hBD-3 in
macrophages
Tumor-derived factors in the tumor microenvironment can
stimulate macrophages to produce a wide array of tumor-
promoting molecules, such as chemokines, cytokines, and growth
factors, to stimulate tumor cell proliferation, tumor angiogenesis
and metastasis [12,32]. To determine whether hBD-3 influences
macrophages to produce tumor-promoting factors, we treated
Figure 4. Xenograft tumors established with parent and hBD-3 overexpressing HEK293 cells in nude mice. (A) RT-PCR of hBD-3 on total
RNA samples extracted from parent HEK293 and hBD-3 overexpressed HEK293 cells, respectively. (B and C) ELISA of hBD-3 using culture supernatants
(B) or cell lysates (C) derived from parent HEK293 and hBD-3 overexpressed cells. HEK293 and hBD-3 overexpressing cells were cultured in serum-free
medium for 3 days, followed by ELISA of collected media and cell lysates, respectively. *, p= 0.00. (D) Representative mice bearing tumors after 10
days post inoculation. Yellow arrows, inoculation sites. (E) Representative tumors isolated from mice inoculated with parent HEK293 cells and hBD-3
overexpressing cells. (F) The incidence and sizes of xenograft tumors generated using parent HEK293 and hBD-3 overexpressed HEK293 cells. The
mean volume for each group of tumors is represented as black lines; in HEK293 tumors, the value is 27.9 mm
3
, while in hBD-3 overexpressed tumors,
the value is 66.9 mm
3
.*,p,0.05.
doi:10.1371/journal.pone.0010993.g004
hBD-3 Regulates TAM via CCR2
PLoS ONE | www.plosone.org 6 June 2010 | Volume 5 | Issue 6 | e10993

Figure 5. Characterizations of xenograft tumors in nude mice inoculated with parent and hBD-3 overexpressing tumorigenic cells.
(A) H&E staining of xenograft tumor sections derived from parent and hBD-3 overexpressing HEK293 cells. (B) Histological features of the xenograft
tumor established from hBD-3 overexpressing HEK293 cells. (a) cells under mitosis are indicated with arrows. Arrowheads indicate fibrous septae, (b)
fibrous septae (arrowhead) divide the nodule into lobules of cell clusters arranged in an organoid pattern (arrow), (c) possible necrotic regions are
indicated with arrowheads. (C) F4/80 (green) images of mouse macrophages in tumor sections derived from parent and hBD-3 overexpressing cells.
Several F4/80+cells are indicated by arrows in the hBD-3 overexpressed section (enlarged inset). Nuclei, blue (DAPI). (D) Double-immunofluorescent
staining of F4/80 (green) and mouse CCR2 (red) in the hBD-3 overexpressed xenograft tumor section. Arrows indicate cells that express both CCR2
and F4/80 in the merged panel. (E) Double-immunofluorescent staining of F4/80 (green) and mouse CCR2 (red) in the parent HEK293 tumor section.
Nuclei, blue (DAPI). (F) HBD-3 (red) in xenograft tumors generated from parent HEK293 (left panel) and hBD-3 overexpressing cells (right panel).
Nuclei, blue (DAPI). Representative images from 2 independent experiments are shown.
doi:10.1371/journal.pone.0010993.g005
hBD-3 Regulates TAM via CCR2
PLoS ONE | www.plosone.org 7 June 2010 | Volume 5 | Issue 6 | e10993

macrophages, which were differentiated from THP-1 monocytic
cells in vitro using phorbol-myristate acetate (Figure 6A), with
recombinant or synthetic hBD-3, followed by real-time quantita-
tive RT-PCR (qPCR) analysis to assess mRNA levels of respective
cytokines and chemokines. The results showed that hBD-3
induced the expression of IL-1a, IL-6, IL-8, and CCL18
(Figure 6C), suggesting that hBD-3 may activate macrophages
and stimulate their tumor-promoting capacity. However, hBD-3
did not significantly induce TNFaexpression in macrophages
differentiated from THP-1 cells (Figure 6C). To determine
whether human macrophages respond to hBD-3, we treated
macrophages, which were differentiated from human peripheral
blood monocytes (PBMs) in vitro with macrophage-colony
stimulating factor (M-CSF) stimulation [33] (Figure 6B), with
hBD-3 and subsequently extracted total RNA for qPCR analysis
of the cytokine/chemokine transcripts. The results indicated that
hBD-3 significantly induced expression of the cytokines/chemo-
kines, including TNFa(Figure 6D). We also performed RT-PCR
analysis using primers specific for IL-10, IL-17, and IL-23,
cytokines that are involved in the tumor-related inflammation
[12,32]. However, the expression of these cytokines was not
detected in macrophages with or without treatment (data not
shown).
Involvement of CCR2 in monocyte/macrophage
migration in response to hBD-3
We have previously shown that synthetic and recombinant
hBD-3 induces migration of THP-1 cells comparably to MCP-1
[19]. In this report, we demonstrated that hBD-3 induced
migration of THP-1 cells in a dose-dependent manner
(Figure 7A). To further examine the chemotactic properties of
hBD-3 to monocytic cells, we performed migration assays using
human PBMs and the monocytic cell line Mono-Mac-1 in
response to hBD-3. Migration data showed that hBD-3 induced
cell migration of PBMs and Mono-Mac-1 cells (Figure 7B). Mono-
Mac-1 was established from peripheral blood of a patient with
monoblastic leukemia [34]. These cells retain distinct morpholog-
ical, cytochemical, and immunological properties of monocytes
[34]. Mono-Mac-1 cells express the chemokine receptor CCR2
and have been used to study monocytic functions in vitro, including
Figure 6. HBD-3 induced cytokines macrophages. (A and B) Macrophages differentiated from THP-1 cells using PMA stimulation (A) and from
peripheral blood monocytes (PBMs) using macrophage-colony stimulating factor (M-CSF) treatment (B) [65]. Note macrophage-like morphological
changes after differentiation. (C) Real-time quantitative RT-PCR of IL-1a, IL-6, IL-8, CCL18, and TNF-ain THP-1 cell-differentiated macrophages treated
with hBD-3 (10 mg/ml) for 16 h. (D) Real-time quantitative RT-PCR of IL-1a, IL-6, IL-8, CCL18, and TNF-ain PBM-differentiated macrophages treated
with hBD-3 (10 mg/ml) for 16 h. Experiments were repeated 3 times. pvalues are presented in each graph.
doi:10.1371/journal.pone.0010993.g006
hBD-3 Regulates TAM via CCR2
PLoS ONE | www.plosone.org 8 June 2010 | Volume 5 | Issue 6 | e10993

chemotaxis [34,35,36]. CCR2 has been shown to play a
nonredundant role as a major mediator of macrophage recruit-
ment via MCP-1 [30,37]. Because we have observed association of
CCR2+macrophage trafficking with tumor cell-produced hBD-3,
but not MCP-1, in oral CIS lesions (Figure 2B, 2C; Figure 3B–D)
and in xenograft tumors in nude mice (Figure 5C–E), we
hypothesized that hBD-3 mediates monocyte/macrophage migra-
tion by acting through CCR2. Cross-desensitization of THP-1
monocytic cells by pretreatment with MCP-1 attenuated cell
migration induced by hBD-3 and similarly, hBD-3 pretreatment
blocked MCP-1 induced cell migration, suggesting that both hBD-
3 and MCP-1 chemoattract monocytic cells through the same
receptor, i.e., CCR2 (Figure 7C). To further confirm the
importance of CCR2 in monocytic cell migration in response to
hBD-3, we treated THP-1 and Mono-Mac-1 cells with the potent
and selective CCR2 pharmacological inhibitor RS102895 [38],
followed by in vitro migration assays in response to hBD-3, MCP-1,
and SDF-1a, respectively. The CCR2 inhibitor blocked THP-1
and Mono-Mac-1 cell migration in response to hBD-3 and MCP-
1, but not SDF-1a(Figure 7D and 7E). Collectively, these data
indicate that hBD-3 induces monocyte/macrophage cell migration
via CCR2.
Discussion
Role of hBD-3 in TAM trafficking
An oral CIS lesion is a histopathologic entity in which dysplastic
cells, arising from the basal layer, occupy the full thickness of the
epithelium from the basement membrane to the surface and in all
likelihood progress to invasive carcinoma [39,40]. We have
previously demonstrated the spatiotemporal expression of hBDs
at various stages of oral cancer and the possible role of hBD-3 in
mediating the tumor-related inflammatory process [19]. In the
present study, our results indicate that tumor cells within CIS
lesions exclusively produce hBD-3, thereby generating the hBD-3-
rich tumor microenvironment. The change in the expression
pattern of hBDs between normal epithelium and the CIS lesion is
probably related to the development and progression of oral
cancer, since the hBD-3 expressing tumor cells are correlated with
the accumulation of tumor-promoting TAMs in the lesion [19].
Figure 7. CCR2 mediated monocytic cell migration in response to hBD-3. (A) Dose-response of THP-1 cell migration in response to hBD-3.
(B) Migration of PBMs and Mono-Mac-1 cells in response to hBD-3 (200 ng/ml) and MCP-1 (30 ng/ml). Migration of PBMs was determined by counting
PBMs in 4 fields under a microscope in each lower chamber (Y-axis on left). To quantify Mono-Mac-1 migration, cells were collected from the lower
chamber of transwell plates, centrifuged and suspended in PBS to count the total number of cells using a hemocytometer (Y-axis on right). cont, no
chemoattractant control; *, p,0.05. (C) Effect of cross-desensitization on THP-1 monocytic cell migration in response to hBD-3 and MCP-1. Cells were
desensitized by pretreatment with 10 mg/ml hBD-3 (hBD3 pretreat) or 100 ng/ml MCP-1 (MCP1 pretreat) for 1 h, followed by migration assays in
response to MCP-1 (30 ng/ml) or hBD-3 (200 ng/ml). Results are representative of 3 independent experiments. *, p,0.05. (D and E) Effect of RS102895
on THP-1 (D) and Mono-Mac-1 (E) monocytic cell migration in response to hBD-3, MCP-1, and SDF-1a(control). Cells were pretreated with RS102895
at 20 mM for 2 h, followed by cell migration assays. hBD-3, 200 ng/ml; MCP-1, 30 ng/ml; SDF-1a, 50 ng/ml. cont, no chemoattractant control; * and **,
p,0.05. THP-1 cell migration was calculated as migration indexes, while Mono-Mac-1 cell migration was quantified as the number of migrated cells.
doi:10.1371/journal.pone.0010993.g007
hBD-3 Regulates TAM via CCR2
PLoS ONE | www.plosone.org 9 June 2010 | Volume 5 | Issue 6 | e10993

TAMs, derived from circulating monocytes, often make up a
significant part of infiltrating immune cells in the tumor
microenvironment and participate in the development and
progression of tumors [30]. Clinical and experimental studies
have shown that TAMs are frequently associated with poor
prognosis in breast, prostate, bladder, and cervical cancers
[2,41,42,43,44,45]. Tumor and stromal cell produced chemokines,
particularly MCP-1, have been associated with recruitment and
infiltration of leukocytes to tumor sites [30]. Growth factors and
cytokines are also described as chemotactic for monocytes/
macrophages during tumor development [7] (Table 1). In oral
squamous cell carcinomas, macrophage infiltration into and
around cancer tissues is significantly correlated with tumor size,
aggressiveness, invasion, and poor prognosis, while infiltrating T
lymphocytes do not correlate with tumor progression [15,16,17].
However, tumor cell-derived molecules that recruit inflammatory
cells to the oral cancer lesion are still largely undetermined
[14,18].
In the current report, we provide novel information about the
role of hBD-3 in regulation of macrophage infiltration into tumors
in vivo. Our data reveal that hBD-3 overexpressing cells, derived
from the tumorigenic HEK293 cell line, form xenograft tumors in
nude mice with massive infiltration of host macrophages compared
with those generated from parent cells. HEK293 is an immortal-
ized cell line established from the sheared adenovirus 5 DNA
transformation of human embryonic kidney cells [46]. The cell
line is tumorigenic and has been used as a tumor model for in vitro
and in vivo assays of transformation, tumor progression, angiogen-
esis, and drug development [47,48,49,50]. Gene expression
profiles of the HEK 293 cell line documented by cDNA expression
microarray analysis have identified low level expression of
chemokines, cytokines, and growth factors under normal culture
conditions [51,52,53], suggesting that HEK293 cell-derived
molecules are unlikely to be involved in attracting host
macrophages in nude mice. Thus, our findings indicate that
hBD-3 is sufficient to induce migration of monocytes/macrophag-
es in vivo and that hBD-3 exhibits a causal relationship with tumor
infiltration of macrophages. It has been described that TAMs
accumulate preferentially in the poorly vascularized, necrotic
regions of tumors that are characterized by low oxygen tension [5].
Our results also revealed that accumulation of TAMs was
particularly associated with necrotic regions of the xenograft
tumors formed by hBD-3 overexpressing HEK293 cells.
HBD-3 and tumorigenicity
Tumorigenicity of HEK293 cells is low when inoculated into
nude mice and ectopic expression of oncogenic molecules, such as
pituitary tumor transforming gene (PTTG), enhances oncogenic
potential of transfected cells as shown by a higher incidence of
tumor formation and rates of growth [54]. Our results showed that
hBD-3 expressing HEK293 cells, but not parent cells, significantly
increased the incidence and growth rates of xenograft tumors in
nude mice, probably through infiltrating host macrophages.
TAMs produce a wide array of tumor-promoting molecules, such
as chemokines, cytokines, and growth factors, in the tumor
microenvironment, to stimulate tumor cell proliferation, tumor
angiogenesis, and metastasis [32]. For example, Lewis lung cancer
(LLC) cells produce the extracellular matrix proteoglycan versican,
which activates local macrophages to induce TNF-asecretion and
subsequently stimulate LLC metastatic growth in vivo. This
suggests that cancer cells can use components of the host innate
immune system, such as TAMs, to generate a prevailing
inflammatory microenvironment for metastasis [12]. In the
current study, we demonstrate that hBD-3 can promote
macrophage expression of IL-1a, IL-6, IL-8, and CCL18; i.e.,
cytokines and chemokines that are produced by TAMs as
components of the cancer-related inflammation [5,55]. HBD-3
also stimulates PBM-derived macrophages to produce TNFa. The
proinflammatory and immunoregulatory cytokine IL-1 has been
shown to play an important role in tumor angiogenesis [56].
Carmi et al. have demonstrated that macrophage-derived IL-1
activates infiltrating myeloid cells to produce VEGF, thus inducing
endothelial cell migration, proliferation and organization into
blood vessel-like structures and promoting tumor angiogenesis
[57]. In their Matrigel plug system, Carmi and colleagues have
shown that neutralization of IL-1 completely abrogated cell
infiltration and angiogenesis and significantly reduced VEGF
levels, thus inducing endothelial cell migration, proliferation and
organization into blood vessel-like structures and promoting tumor
angiogenesis [57]. IL-6 is a potent inflammatory cytokine that is
considered a key tumor-promoting and antiapoptotic factor
[58,59]. IL-6 contributes to the induction of skin tumors [58],
triggers malignant features in breast tumor mammospheres [60],
and participates in suppression of antigen-specific anti-tumor
immunity through up-regulation of macrophage B7-H4 expression
[61]. TAM expression of IL-8 and a number of molecules, such as
VEGF and TNF-a, have been implicated in enhanced angiogen-
esis [5,62], while TAM-produced CCL18 can recruit naı
¨ve T cells
to the microenvironment dominated by TAMs for possible T cell
anergy [5]. Therefore, the tumorigenic effect of hBD-3 on
xenograft tumor growth suggests that the inflammatory cells and
molecules in the tumor microenvironment can affect a variety of
transcriptional programs to promote tumor development and
growth. Clearly, direct hBD-3 modulation of effector functions of
TAMs in vivo needs to be further elucidated.
CCR2 and monocytic cell migration in response to hBD-3
We and others have demonstrated that hBD-3 chemoattracts
monocytes, including cells of monocytic cell lines and peripheral
blood monocytes, in vitro [19,28,29]. The chemokine receptor
CCR6 has been identified to mediate memory T cell and iDC
migration in response to hBD-1 and hBD-2 [27]. HBD-3 has also
been shown to be chemotactic for HEK293 cells overexpressing
CCR6 [28]. However, the receptor that hBD-3 interacts with to
recruit monocytic cells has not been identified, since these cells do
not express CCR6 [28]. Here, we provide novel evidence that
hBD-3 chemoattracts monocytic cells by acting through the
chemokine receptor CCR2. Our cross-desensitization and the
CCR2 inhibitor results indicate that hBD-3 and MCP-1
chemoattract monocytes by acting via the same CCR2 receptor.
MCP-1 is a potent chemoattractant for monocytes, DCs, and
natural killer (NK) cells [63]. MCP-1 interacts with the chemokine
receptor CCR2 and triggers decoupling of G
i
-derived asubunit
from Gbc [64]. Activation of the CCR2 signaling pathway by
MCP-1 initiates cascades of specific intracellular signaling events,
including activation of phosphoinositide 3-kinases (PI3K) and
phospholipase Cb(PLCb), intracellular calcium mobilization, and
activation of PKC and ERK, resulting in cell migration [64].
RS102895 is a member of the spiropiperidine molecule class with
potent and specific inhibitory properties to CCR2b, a splicing
variant of the CCR2 gene that has higher binding affinity to MCP-
1 and mediates chemoattraction and intracellular calcium influx
by MCP-1 [36,38,64]. The binding of RS102895 to CCR2b
blocks receptor binding of MCP-1, subsequently inhibiting
intracellular calcium influx, cAMP inhibition, and chemotaxis by
MCP-1 [38]. Our results suggest that hBD-3 may interact directly
with CCR2 and subsequently activate its signaling pathways to
induce monocyte migration. The notion is supported by recent
hBD-3 Regulates TAM via CCR2
PLoS ONE | www.plosone.org 10 June 2010 | Volume 5 | Issue 6 | e10993

work in which HEK293 cells that overexpress CCR2B migrate to
the hBD-3:IgG fusion protein. In addition, mouse peritoneal
exudate cells (PECs) derived from CCR2 deficient (Ccr2
2/2
)
C57BL/6 mice fail to migrate in response to hBD-3 (Joost
Oppenheim; personal communication). Collectively, these results
support our hypothesis that hBD-3 functions as a chemoattractant
to recruit macrophages and that CCR2 plays a central role
in mediating monocyte/macrophage migration in response to
hBD-3.
In conclusion, we demonstrate herein that tumor cell-produced
hBD-3 functions as a chemoattractant for recruitment of TAMs in
the development of tumors and that hBD-3 chemoattracts
monocytes/macrophages via the chemokine receptor CCR2. This
novel mechanism is the first evidence of an hBD molecule
orchestrating an in vivo outcome and demonstrates its importance
in establishing a tumor-associated inflammatory microenviron-
ment, which supports growth and progression of tumors.
Materials and Methods
Ethics statement
Tissue sample protocols for samples obtained from the
Department of Oral Pathology, Case Western Reserve University
School of Dental Medicine, and waiver of informed consent were
approved by Case Cancer Institutional Review Board. All animal
experiments were conducted in compliance with the Cleveland
State University Institutional Animal Care and Use Committee.
Written informed consents and protocols using human blood were
approved by the Cleveland Clinic Institutional Review Board.
Cell culture and reagents
THP-1 and HEK293 cells were obtained from American Type
Culture Collection (Manassas, VA) and maintained in
RPMI1640/10% FBS (Innovative Res., Novi, MI) and in
DMEM/10% FBS, respectively. Mono-Mac-1 cells were provided
by Dr. Sabina Sperandio (Centre de Recherche du CHUL,
Canada) and cultured in RPMI1640/10% FBS. Differentiation of
macrophages from THP-1 cells was performed as described by
Tjiu et al [65]. Peripheral blood monocytes were prepared from
human blood as previously described [66] and were differentiated
to macrophages as described [33]. Recombinant hBD-3 was
produced and tested for endotoxin contamination as we described
previously [25]. Synthetic hBD-3 was purchased from Peptide
International (Louisville, KY). SDF-1aand MCP-1 were pur-
chased from PeproTech (Rocky Hill, NJ). Antibodies used in our
studies were: goat polyclonal anti-hBD-2, goat polyclonal anti-
MCP-1, mouse monoclonal anti-CCR2 (for human tissue), and rat
monoclonal anti-F4/80 (Santa Cruz Biotech., Santa Cruz, CA);
rabbit anti-mouse CCR2 (Abcam, Cambridge, MA); rabbit
polyclonal anti-hBD-3 and mouse monoclonal anti-CD68 (Novus,
Littleton, CO); AlexaFluor488-conjugated donkey antibodies to
IgGs of mouse, goat, and rat as well as AlexFluor594-conjugated
donkey antibodies to IgGs of rabbit and mouse (Invitrogen,
Carlsbad, CA). Chromatographically purified IgGs of rabbit,
mouse, goat, and rat were purchased from Invitrogen. Phorbol 12-
myristate-13-acetate and RS10289 5 was purchased from Sigma-
Aldrich (St Louis, MO) and dissolved in DMSO as stocks.
Immunofluorescence microscopy
Formalin-fixed, paraffin-embedded biopsy specimens were
obtained from the Department of Oral Pathology, Case Western
Reserve University School of Dental Medicine. We previously
described methods for immunofluorescence microscopy [19].
Briefly, each section (5 mm) was de-paraffinized in xylene and
hydrated with serially diluted ethanol, followed by blocking with
10% donkey serum overnight at 4uC. After washing with PBS,
each section was incubated with the respective primary antibody
(1 h, room temperature), washed in PBS (3610 min), and then
stained with the compatible fluoresce dye-conjugated secondary
antibody. For double immunofluorescence, consecutive staining by
different primary and secondary antibodies was performed.
Isotype controls were conducted using isotype-matched IgGs,
corresponding to each primary antibody. Sections were mounted
on slides with the VECTASHIELD Fluorescent Mounting Media
(Vector Lab Inc., Burlingame, CA) containing DAPI to visualize
nuclei. Immunofluorescent images were generated using a Leica
DMI 6000B fluorescence microscope (Leica Microsystems,
Bannockburn, IL) or an Olympus BX51 fluorescence microscope
mounted with the Olympus DP71 camera (Olympus America Inc.,
Center Valley, PA). Immunofluorescence images were processed
using the NIH ImageJ program [67]. To quantify expression levels
of hBD-3, normal and CIS immunofluorescent images of hBD-2
and hBD-3 were acquired in 16-bit gray scale, respectively.
Fluorescent densities on each of the antibody treated sections were
measured with the NIH ImageJ program as described previously
[19,67]. The expression of hBD-2 and hBD-3 was represented as
the ratio of relative fluorescence intensity of hBD-2 and hBD-3
over that of nuclei, respectively.
Chemotaxis assay and ELISA for hBD-3
Chemotaxis assays were performed as described previously [19].
Briefly, serum-free RPMI1640 media containing hBD-3, MCP-1,
or SDF-1awere added to each lower well of the Millicell-24 plate
assemblies (5 mm membrane pore size) (Millipore, Billerica, MA).
Cells (3610
5
in 100 ml of serum-free RPMI1640) were added to
the upper wells. After incubation for 6 h, cells migrating into the
lower-chamber were counted in 4 fields under a microscope (for
PBMs), or collected and counted using a hemocytometer (for
Mono-Mac-1 cells). Each experiment was repeated at least three
times. Chemotactic activity was measured as either migration
index, i.e., the ratio of the number of migrating cells in the lower
well towards a chemoattractant when compared to medium alone,
or the number of cells in the lower wells. The results were
presented as mean 6SD of triplicate wells.
Concentrations of hBD-3 in cell lysates and medium superna-
tants were quantified using an enzyme-linked immunosorbent
assay (ELISA) method as we described previously [68]. Briefly, 96-
well immunoplates (R&D Systems, MN) were coated with 100 ml
anti-hBD-3 antibodies (PeproTech) diluted to 1 mg/ml overnight
at 4uC, followed by blocking with 1% bovine serum albumin (BSA)
in phosphate buffered saline (PBS). Cell lysates, medium
supernatants, and recombinant hBD-2 standards were incubated
at room temperature for 1 h. The wells were washed 3 times with
PBS containing 0.1% Tween 20 and incubated at room
temperature with 100 ml of secondary antibody (PeproTech)
diluted to 0.2 mg/ml, for 30 min. Each plate was washed 3 times
and filled with 50 ml/well streptavidin-peroxidase (Roche Diag-
nostics; 1:10,000 in PBS containing 0.1% Tween 20). Each plate
was then incubated at room temperature for an additional 30 min,
washed 3 times as described above, and incubated with 2,2-azino-
bis-3-ethylbenzthiazoline-6-sulfonic acid (Roche Diagnostics,
Branchburg, NJ) in the dark at room temperature for 20 min.
Absorbance was measured at 415 nm with a microplate reader
(Model 680, Bio-Rad, Hercules, CA).
RT-PCR, and real-time quantitative RT-PCR
Total RNA was extracted using GeneElute mammalian total
RNA isolation kit (Sigma, St. Louise, MO) following the
hBD-3 Regulates TAM via CCR2
PLoS ONE | www.plosone.org 11 June 2010 | Volume 5 | Issue 6 | e10993

manufacture’s protocol as described previously [69]. Briefly, cells
grown in 6-well plates were lysed using the lysis Buffer and the
cellular lysates were centrifuged through the shredding columns.
After collection of total RNA with the RNA column, the column
was washed and the RNA was eluted using RNase-free H
2
O.
Total RNA samples were quantified using a spectrophotometer at
A
260
and samples with the A
260
/A
280
ratio $1.8 were used. For
reverse transcription, RNA (1 mg) was used for the first strand
cDNA synthesis using the SuperScript III reverse-transcriptase
(Invitrogen) in a total volume of 20 ml according to the
manufacturer’s instructions. For RT-PCR analysis, the cDNA
(2 ml) was used in a 25 ml of PCR amplification using Tag DNA
polymerase (Invitrogen) with the following primers: IL-1a:59-
CGCCAATGACTCAGAGGAAGA (forward) and 59-AGGGC-
GTCATTCAGGATGAA (reverse), IL-6: 59-TTCAATGAGGA-
GACTTGCCTG (forward) and 59-ACAACAACAATCTGAG-
GTGCC (reverse), IL-8: 59-GCCAGGAAGAAACCACCGGAA-
GGA (forward) and 59-GGGGTCCAGACAGAGCTCTCT-
TCC, CCL18: 59-CTCCTTGTCCTCGTCTGCAC (forward)
and 59-TCAGGCATTCAGCTTCAGGT (reverse), TNFa:59-
CAGAGGGAAGAGTTCCCCAG (forward) and 59-CCTTGG-
TCTGGTAGGAGACG (reverse), b-actin: 59-GCTCGTCGT-
CGACAACGGCTC (forward) and 59-CAAACATGATCTG-
GGTCATCTTCTC (reverse). For real-time quantitative RT-
PCR (qPCR), total RNA (500 ng) was reverse transcribed using
iScript cDNA synthesis kit (Bio-Rad) following the manufacture’s
protocol. Two ml of the reverse transcription (RT) reaction was
used as a template for real-time PCR using a SYBR Green
Supermix (Bio-Rad) with SYBE green 1 dye as the amplicon
detector according to the manufacture’s protocol. The gene for
glyceraldehyde 3-phosphate dehydrogenase (GAPDHs was ampli-
fied as an endogenous reference. Amplification was performed at
40 cycles of 94uC for 15 s followed by 60uC for 1 min. Primers
used for qPCR are listed in Table 2. Quantification was
determined by using the comparative DDC
T
method as described
by Peinequin et al [70]. Each qPCR was run in triplicates and the
experiment was repeated at least 3 times.
Transfection and mouse model
HBD-3 cDNA was cloned into pcDNA3.1 expression vector
and the cDNA sequence was confirmed through DNA sequencing
performed by the Genomics Core at Lerner Research Institute,
Cleveland Clinic Foundation. Transfection of HEK293 cells was
done with LipofectaminePlus following the manufacturer’s
protocol (Invitrogen). To generate xenograft tumors in nude mice
(nu/nu, National Cancer Institute, Frederick, MD), hBD-3
overexpressing and parent HEK293 cells were trypsinized and
suspended in PBS at 2610
7
cells/ml, respectively. Each animal
was injected (2610
6
cells per injection) subcutaneously in two sites
on opposite sides of the dorsum of the anterior part of the body.
Ten days after inoculation, animals were sacrificed and tumor
incidences were determined by counting tumors that could be
identified visually. Isolated tumors were measured using a caliper
and the tumor volumes were calculated with the equation V
(mm
3
)=(a6b
2
)/2, where ais the largest diameter and bis the
perpendicular diameter in mm [71]. Three tumors from each
group of mice were sectioned and subjected to immunofluores-
cence microscopy with the rat monoclonal antibody to F4/80,
goat polyclonal antibody to mouse CCR2, and rabbit polyclonal
antibody to hBD-3.
Statistics
Results of migration to chemoattractants (migration index or
the number of migrated cells) were compared with respective
controls. The data were subjected to two-tailed paired Student’s t
test with two-sample equal variance for comparison of two groups.
For quantification of immunofluorescence intensity, two-tailed
paired Student’s ttest with two-sample unequal variance was used.
p,0.05 was considered to be statistically significant. Data analyses
were performed and graphs were generated using Minitab
program (Minitab Inc.) and Exel 2003 (Microsoft, Seattle, WA).
Acknowledgments
We thank Dr. Scott Howell (Case Western Reserve University School of
Medicine) for technical assistance on immunofluorescence microscopy, Dr.
Sabina Sperandio (Centre de Recherche du CHUL, Canada) for Mono-
Mac-1 cells, and Drs. Joost Oppenheim and De Yang (National Cancer
Institute, Frederic, MD) for very helpful discussions.
Author Contributions
Conceived and designed the experiments: GJ HIK. Performed the
experiments: GJ HIK CZ XJ SKG AZ. Analyzed the data: GJ HIK
SAH AZ AW. Contributed reagents/materials/analysis tools: HIK SAH
ZF QZ AZ TMM AW. Wrote the paper: GJ. Edited the paper: AW.
References
1. de Visser KE, Eichten A, Coussens LM (2006) Paradoxical roles of the immune
system during cancer development. Nat Rev Cancer 6: 24–37.
2. Leek RD, Lewis CE, Whitehouse R, Greenall M, Clarke J, et al. (1996)
Association of macrophage infiltration with angiogenesis and prognosis in
invasive breast carcinoma. Cancer Res 56: 4625–4629.
3. Ribatti D, Ennas MG, Vacca A, Ferreli F, Nico B, et al. (2003) Tumor
vascularity and tryptase-positive mast cells correlate with a poor prognosis in
melanoma. Eur J Clin Invest 33: 420–425.
4. Imada A, Shijubo N, Kojima H, Abe S (2000) Mast cells correlate with angiogenesis
and poor outcome in stage I lung adenocarcinoma. Eur Respir J 15: 1087–1093.
Table 2. Primers used in real-time quantitative RT-PCR.
Gene names Forward primer (59R39) Reverse primer (59R39) References
IL1-aCGCCAATGACTCAGAGGAAGA AGGGCGTCATTCAGGATGAA [98]
IL-6 GGTACATCCTCGACGGCATCT AGTGCCTCTTTGCTGCTTTCAC [99]
IL-8 CTTGGCAGCCTTCCTGATTT TTCTTTAGCACTCCTTGGCAAAA [98]
CCL18 CTTTCCCCTTTCCCTTCAAC GTGCTGAGCAAAACCATTCA [100]
TNFaTCTTCTCGAACCCCGAGTGA CCTCTGATGGCACCACCAG [98]
GAPDH TCG GAG TCA ACG GAT TT CCA CGA CGT ACT CAG C [101]
doi:10.1371/journal.pone.0010993.t002
hBD-3 Regulates TAM via CCR2
PLoS ONE | www.plosone.org 12 June 2010 | Volume 5 | Issue 6 | e10993

5. Sica A, Allavena P, Mantovani A (2008) Cancer related inflammation: The
macrophage connection. Cancer Lett.
6. Pollard JW (2004) Tumour-educated macrophages promote tumour progres-
sion and metastasis. Nat Rev Cancer 4: 71–78.
7. Allavena P, Sica A, Solinas G, Porta C, Mantovani A (2008) The inflammatory
micro-environment in tumor progression: the role of tumor-associated
macrophages. Crit Rev Oncol Hematol 66: 1–9.
8. Lin EY, Pollard JW (2007) Tumor-associated macrophages press the
angiogenic switch in breast cancer. Cancer Res 67: 5064–5066.
9. Qian B, Deng Y, Im JH, Muschel RJ, Zou Y, et al. (2009) A distinct
macrophage population mediates metastatic breast cancer cell extravasation,
establishment and growth. PLoS One 4: e6562.
10. Lin EY, Nguyen AV, Russell RG, Pollard JW (2001) Colony-stimulating factor
1 promotes progression of mammary tumors to malignancy. J Exp Med 193:
727–740.
11. Zeisberger SM, Odermatt B, Marty C, Zehnder-Fjallman AH, Ballmer-
Hofer K, et al. (2006) Clodronate-liposome-mediated depletion of tumour-
associated macrophages: a new and highly effective antiangiogenic therapy
approach. Br J Cancer 95: 272–281.
12. Kim S, Takahashi H, Lin WW, Descargues P, Grivennikov S, et al. (2009)
Carcinoma-produced factors activate myeloid cells through TLR2 to stimulate
metastasis. Nature 457: 102–106.
13. Mantovani A, Schioppa T, Porta C, Allavena P, Sica A (2006) Role of tumor-
associated macrophages in tumor progression and invasion. Cancer Metastasis
Rev 25: 315–322.
14. Kurago ZB, Lam-Ubol A, Stetsenko A, De La Mater C, Chen Y, et al. (2008)
Lipopolysaccharide-Squamous Cell Carcinoma-Monocyte Interactions Induce
Cancer-Supporting Factors Leading to Rapid STAT3 Activation. Head Neck
Pathol 2: 1–12.
15. Li C, Shintani S, Terakado N, Nakashiro K, Hamakawa H (2002) Infiltration
of tumor-associated macrophages in human oral squamous cell carcinoma.
Oncol Rep 9: 1219–1223.
16. Liu SY, Chang LC, Pan LF, Hung YJ, Lee CH, et al. (2008) Clinicopathologic
significance of tumor cell-lined vessel and microenvironment in oral squamous
cell carcinoma. Oral Oncol 44: 277–285.
17. Marcus B, Arenberg D, Lee J, Kleer C, Chepeha DB, et al. (2004) Prognostic
factors in oral cavity and oropharyngeal squamous cell carcinoma. Cancer 101:
2779–2787.
18. Buettner M, Meyer B, Schreck S, Niedobitek G (2007) Expression of RANTES
and MCP-1 in epithelial cells is regulated via LMP1 and CD40. Int J Cancer
121: 2703–2710.
19. Kawsar HI, Weinberg A, Hirsch SA, Venizelos A, Howell S, et al. (2009)
Overexpression of human b-defensin-3 in oral dysplasia: Potential role in
macrophage trafficking. Oral Oncol 45: 12.
20. Kesting MR, Loeffelbein DJ, Hasler RJ, Wolff KD, Rittig A, et al. (2009)
Expression profile of human b-defensin 3 in oral squamous cell carcinoma.
Cancer Invest 27: 575–581.
21. Harder J, Bartels J, Christophers E, Schroder JM (1997) A peptide antibiotic
from human skin. Nature 387: 861.
22. Harder J, Bartels J, Christophers E, Schroder JM (2001) Isolation and
characterization of human b-defensin-3, a novel human inducible peptide
antibiotic. J Biol Chem 276: 5707–5713.
23. Bensch KW, Raida M, Magert HJ, Schulz-Knappe P, Forssmann WG (1995)
hBD-1: a novel b-defensin from human plasma. FEBS Lett 368: 331–335.
24. Yang D, Liu ZH, Tewary P, Chen Q, de la Rosa G, et al. (2007) Defensin
participation in innate and adaptive immunity. Curr Pharm Des 13:
3131–3139.
25. Feng Z, Dubyak GR, Lederman MM, Weinberg A (2006) Cutting edge:
human bdefensin 3–a novel antagonist of the HIV-1 coreceptor CXCR4.
J Immunol 177: 782–786.
26. Funderburg N, Lederman MM, Feng Z, Drage MG, Jadlowsky J, et al. (2007)
Human -defensin-3 activates professional antigen-presenting cells via Toll-like
receptors 1 and 2. Proc Natl Acad Sci U S A 104: 18631–18635.
27. Yang D, Chertov O, Bykovskaia SN, Chen Q, Buffo MJ, et al. (1999) b-
defensins: linking innate and adaptive immunity through dendritic and T cell
CCR6. Science 286: 525–528.
28. Taylor K, Clarke DJ, McCullough B, Chin W, Seo E, et al. (2008) Analysis and
separation of residues important for the chemoattractant and antimicrobial
activities of b-defensin 3. J Biol Chem 283: 6631–6639.
29. Soruri A, Grigat J, Forssmann U, Riggert J, Zwirner J (2007) b-Defensins
chemoattract macrophages and mast cells but not lymphocytes and dendritic
cells: CCR6 is not involved. Eur J Immunol 37: 2474–2486.
30. Mantovani A, Allavena P, Sozzani S, Vecchi A, Locati M, et al. (2004)
Chemokines in the recruitment and shaping of the leukocyte infiltrate of
tumors. Semin Cancer Biol 14: 155–160.
31. Leenen PJ, de Bruijn MF, Voerman JS, Campbell PA, van Ewijk W (1994)
Markers of mouse macrophage development detected by monoclonal
antibodies. J Immunol Methods 174: 5–19.
32. Mantovani A, Allavena P, Sica A, Balkwill F (2008) Cancer-related
inflammation. Nature 454: 436–444.
33. Hacker G, Redecke V, Hacker H (2002) Activation of the immune system by
bacterial CpG-DNA. Immunology 105: 245–251.
34. Steube KG, Teepe D, Meyer C, Zaborski M, Drexler HG (1997) A model
system in haematology and immunology: the human monocytic cell line
MONO-MAC-1. Leuk Res 21: 327–335.
35. Mellado M, Rodriguez-Frade JM, Aragay A, del Real G, Martin AM, et al.
(1998) The chemokine monocyte chemotactic protein 1 triggers Janus kinase 2
activation and tyrosine phosphorylation of the CCR2B receptor. J Immunol
161: 805–813.
36. Aragay AM, Mellado M, Frade JM, Martin AM, Jimenez-Sainz MC, et al.
(1998) Monocyte chemoattractant protein-1-induced CCR2B receptor desen-
sitization mediated by the G protein-coupled receptor kinase 2. Proc Natl Acad
Sci U S A 95: 2985–2990.
37. Zhou Y, Yang Y, Warr G, Bravo R (1999) LPS down-regulates the expression
of chemokine receptor CCR2 in mice and abolishes macrophage infiltration in
acute inflammation. J Leukoc Biol 65: 265–269.
38. Mirzadegan T, Diehl F, Ebi B, Bhakta S, Polsky I, et al. (2000) Identification of
the binding site for a novel class of CCR2b chemokine receptor antagonists:
binding to a common chemokine receptor motif within the helical bundle. J Biol
Chem 275: 25562–25571.
39. Waldron CA, Shafer WG (1975) Oral carcinoma in situ. Oral Surg Oral Med
Oral Pathol 39: 227–238.
40. Sciubba JJ (2001) Oral cancer. The importance of early diagnosis and
treatment. Am J Clin Dermatol 2: 239–251.
41. Himes RH, Burton PR, Kersey RN, Pierson GB (1976) Brain tubulin
polymerization in the absence of ‘‘microtubule-associated proteins’’. Proc Natl
Acad Sci U S A 73: 4397–4399.
42. Lissbrant IF, Stattin P, Wikstrom P, Damber JE, Egevad L, et al. (2000) Tumor
associated macrophages in human prostate cancer: relation to clinicopatho-
logical variables and survival. Int J Oncol 17: 445–451.
43. Hanada T, Nakagawa M, Emoto A, Nomura T, Nasu N, et al. (2000)
Prognostic value of tumor-associated macrophage count in human bladder
cancer. Int J Urol 7: 263–269.
44. Salvesen HB, Akslen LA (1999) Significance of tumour-associated macrophag-
es, vascular endothelial growth factor and thrombospondin-1 expression for
tumour angiogenesis and prognosis in endometrial carcinomas. Int J Canc er
84: 538–543.
45. Fujimoto J, Sakaguchi H, Aoki I, Tamaya T (2000) Clinical implications of
expression of interleukin 8 related to angiogenesis in uterine cervical cancers.
Cancer Res 60: 2632–2635.
46. Graham FL, Smiley J, Russell WC, Nairn R (1977) Characteristics of a human
cell line transformed by DNA from human adenovirus type 5. J Gen Virol 36:
59–74.
47. Papageorgiou A, Lashinger L, Millikan R, Grossman HB, Benedict W, et al.
(2004) Role of tumor necrosis factor-related apoptosis-inducing ligand in
interferon-induced apoptosis in human bladder cancer cells. Cancer Res 64:
8973–8979.
48. Shen C, Gu M, Song C, Miao L, Hu L, et al. (2008) The tumorigenicity
diversification in human embryonic kidney 293 cell line cultured in vitro.
Biologicals 36: 263–268.
49. Chao C, Goluszko E, Lee YT, Kolokoltsov AA, Davey RA, et al. (2007)
Constitutively active CCK2 receptor splice variant increases Src-dependent
HIF-1 aexpression and tumor growth. Oncogene 26: 1013–1019.
50. Kessler O, Shraga-Heled N, Lange T, Gutmann-Raviv N, Sabo E, et al. (2004)
Semaphorin-3F is an inhibitor of tumor angiogenesis. Cancer Res 64:
1008–1015.
51. Li J, Chen X, Gong X, Liu Y, Feng H, et al. (2009) A transcript profiling
approach reveals the zinc finger transcription factor ZNF191 is a pleiotropic
factor. BMC Genomics 10: 241.
52. Zheng XH, Watts GS, Vaught S, Gandolfi AJ (2003) Low-level arsenite
induced gene expression in HEK293 cells. Toxicology 187: 39–48.
53. Satoh J, Yamamura T (2004) Gene expression profile following stable
expression of the cellular prion protein. Cell Mol Neurobiol 24: 793–814.
54. Malik MT, Kakar SS (2006) Regulation of angiogenesis and invasion by
human Pituitary tumor transforming gene (PTTG) through increased
expression and secretion of matrix metalloproteinase-2 (MMP-2). Mol Cancer
5: 61.
55. Burnett GT, Weathersby DC, Taylor TE, Bremner TA (2008) Regulation of
inflammation- and angiogenesis-related gene expression in breast cancer cells
and co-cultured macrophages. Anticancer Res 28: 2093–2099.
56. Voronov E, Carmi Y, Apte RN (2007) Role of IL-1-mediated inflammation in
tumor angiogenesis. Adv Exp Med Biol 601: 265–270.
57. Carmi Y, Voronov E, Dotan S, Lahat N, Rahat MA, et al. (2009) The role of
macrophage-derived IL-1 in induction and maintenance of angiogenesis.
J Immunol 183: 4705–4714.
58. Dranoff G (2004) Cytokines in cancer pathogenesis and cancer therapy. Nat
Rev Cancer 4: 11–22.
59. Lin WW, Karin M (2007) A cytokine-mediated link between innate immunity,
inflammation, and cancer. J Clin Invest 117: 1175–1183.
60. Sansone P, Storci G, Tavolari S, Guarnieri T, Giovannini C, et al. (2007) IL-6
triggers malignant features in mammospheres from human ductal breast
carcinoma and normal mammary gland. J Clin Invest 117: 3988–4002.
61. Kryczek I, Zou L, Rodriguez P, Zhu G, Wei S, et al. (2006) B7-H4 expression
identifies a novel suppressive macrophage population in human ovarian
carcinoma. J Exp Med 203: 871–881.
hBD-3 Regulates TAM via CCR2
PLoS ONE | www.plosone.org 13 June 2010 | Volume 5 | Issue 6 | e10993

62. Cramer T, Yamanishi Y, Clausen BE, Forster I, Pawlinski R, et al. (2003) HIF-
1ais essential for myeloid cell-mediated inflammation. Cell 112: 645–657.
63. Conti I, Rollins BJ (2004) CCL2 (monocyte chemoattractant protein-1) and
cancer. Semin Cancer Biol 14: 149–154.
64. Jimenez-Sainz MC, Fast B, Mayor F, Jr., Aragay AM (2003) Signaling
pathways for monocyte chemoattractant protein 1-mediated extracellular
signal-regulated kinase activation. Mol Pharmacol 64: 773–782.
65. Tjiu JW, Chen JS, Shun CT, Lin SJ, Liao YH, et al. (2009) Tumor-associated
macrophage-induced invasion and angiogenesis of human basal cell carcinoma
cells by cyclooxygenase-2 induction. J Invest Dermatol 129: 1016–1025.
66. Shashkin PN, Jain N, Miller YI, Rissing BA, Huo Y, et al. (2006) Insulin and
glucose play a role in foam cell formation and function. Cardiovasc Diabetol 5:
13.
67. Collins TJ (2007) ImageJ for microscopy. Biotechniques 43: 25–30.
68. Ghosh SK, Gerken TA, Schneider KM, Feng Z, McCormick TS, et al. (2007)
Quantification of human b-defensin-2 and -3 in body fluids: application for
studies of innate immunity. Clin Chem 53: 757–765.
69. Kawsar HI, Ghosh SK, Hirsch SA, Koon HB, Weinberg A, et al. (2010)
Expression of human b-defensin-2 in intratumoral vascular endothelium and in
endothelial cells induced by transforming growth factor b. Peptides 31: 7.
70. Peinnequin A, Mouret C, Birot O, Alonso A, Mathieu J, et al. (2004) Rat pro-
inflammatory cytokine and cytokine related mRNA quantification by real-time
polymerase chain reaction using SYBR green. BMC Immunol 5: 3.
71. Cantor JP, Iliopoulos D, Rao AS, Druck T, Semba S, et al. (2007) Epigenetic
modulation of endogenous tumor suppres sor expression in lung cancer
xenografts suppresses tumorigenicity. Int J Cancer 120: 24–31.
72. Payne AS, Cornelius LA (2002) The role of chemokines in melanoma tumor
growth and metastasis. J Invest Dermatol 118: 915–922.
73. Fujimoto H, Sangai T, Ishii G, Ikehara A, Nagashima T, et al. (2009) Stromal
MCP-1 in mammary tumors induces tumor-associated macrophage infiltration
and contributes to tumor progression. Int J Cancer 125: 1276–1284.
74. Wu Y, Li YY, Matsushima K, Baba T, Mukaida N (2008) CCL3-CCR5 axis
regulates intratumoral accumulation of leukocytes and fibroblasts and promotes
angiogenesis in murine lung metastasis process. J Immunol 181: 6384–6393.
75. Erreni M, Bianchi P, Laghi L, Mirolo M, Fabbri M, et al. (2009) Expression of
chemokines and chemokine receptors in human colon cancer. Methods
Enzymol 460: 105–121.
76. Zucchetto A, Benedetti D, Tripodo C, Bomben R, Dal Bo M, et al. (2009)
CD38/CD31, the CCL3 and CCL4 chemokines, and CD49d/vascular cell
adhesion molecule-1 are interchained by sequential events sustaining chronic
lymphocytic leukemia cell survival. Cancer Res 69: 4001–4009.
77. Lentzsch S, Gries M, Janz M, Bargou R, Dorken B, et al. (2003) Macrophage
inflammatory protein 1-a(MIP-1 a) triggers migration and signaling cascades
mediating survival and proliferation in multiple myeloma (MM) cells. Blood
101: 3568–3573.
78. Soria G, Ben-Baruch A (2008) The inflammatory chemokines CCL2 and
CCL5 in breast cancer. Cancer Lett 267: 271–285.
79. Ben-Baruch A (2006) The multifaceted roles of chemokines in malignancy.
Cancer Metastasis Rev 25: 357–371.
80. Mrowietz U, Schwenk U, Maune S, Bartels J, Kupper M, et al. (1999) The
chemokine RANTES is secreted by human melanoma cells and is associated
with enhanced tumour formation in nude mice. Br J Cancer 79: 1025–1031.
81. Behbahani H, Walther-Jallow L, Klareskog E, Baum L, French AL, et al.
(2007) Proinflammatory and type 1 cytokine expression in cervical mucosa
during HIV-1 and human papillomavirus infection. J Acquir Immune Defic
Syndr 45: 9–19.
82. Niwa Y, Akamatsu H, Niwa H, Sumi H, Ozaki Y, et al. (2001) Correlation of
tissue and plasma RANTES levels with disease course in patients with breast or
cervical cancer. Clin Cancer Res 7: 285–289.
83. Schioppa T, Uranchimeg B, Saccani A, Biswas SK, Doni A, et al. (2003)
Regulation of the chemokine receptor CXCR4 by hypoxia. J Exp Med 198:
1391–1402.
84. Joyce JA, Pollard JW (2009) Microenvironmental regulation of metastasis. Nat
Rev Cancer 9: 239–252.
85. Bottazzi B, Erba E, Nobili N, Fazioli F, Rambaldi A, et al. (1990) A paracrine
circuit in the regulation of the proliferation of macrophages infiltrating murine
sarcomas. J Immunol 144: 2409–2412.
86. Byrne PV, Guilbert LJ, Stanley ER (1981) Distribution of cells bearing
receptors for a colony-stimulating factor (CSF-1) in murine tissues. J Cell Biol
91: 848–853.
87. Fu YX, Watson G, Jimenez JJ, Wang Y, Lopez DM (1990) Expansion of
immunoregulatory macrophages by granulocyte-macrophage colony-stimulat-
ing factor derived from a murine mammary tumor. Cancer Res 50: 227–234.
88. Dougherty ST, Eaves CJ, McBride WH, Dougherty GJ (1997) Role of
macrophage-colony-stimulating factor in regulating the accumulation and
phenotype of tumor-associated macrophages. Cancer Immunol Immunother
44: 165–172.
89. Hansen G, Hercus TR, McClure BJ, Stomski FC, Dottore M, et al. (2008) The
structure of the GM-CSF receptor complex reveals a distinct mode of cytokine
receptor activation. Cell 134: 496–507.
90. Hiratsuka S, Nakamura K, Iwai S, Murakami M, Itoh T, et al. (2002) MMP9
induction by vascular endothelial growth factor receptor-1 is involved in lung-
specific metastasis. Cancer Cell 2: 289–300.
91. Hiratsuka S, Watanabe A, Aburatani H, Maru Y (2006) Tumour-mediated
upregulation of chemoattractants and recruitment of myeloid cells predeter-
mines lung metastasis. Nat Cell Biol 8: 1369–1375.
92. Lewis JS, Landers RJ, Underwood JC, Harris AL, Lewis CE (2000) Expression
of vascular endothelial growth factor by macrophages is up-regulated in poorly
vascularized areas of breast carcinomas. J Pathol 192: 150–158.
93. Wendt MK, Schiemann WP (2009) Therapeutic targeting of the focal adhesion
complex prevents oncogenic TGF-bsignaling and metastasis. Breast Cancer
Res 11: R68.
94. Shabo I, Stal O, Olsson H, Dore S, Svanvik J (2008) Breast cancer expression
of CD163, a macrophage scavenger receptor, is related to early distant
recurrence and reduced patient survival. Int J Cancer 123: 780–786.
95. Markiewski MM, Lambris JD (2009) Unwelcome complement. Cancer Res 69:
6367–6370.
96. Markiewski MM, Lambris JD (2009) Is complement good or bad for cancer
patients? A new perspective on an old dilemma. Trends Immunol 30: 286–292.
97. Mantovani A, Sica A (2010) Macrophages, innate immunity and cancer:
balance, tolerance, and diversity. Curr Opin Immunol 22: 231–237.
98. Overbergh L, Kyama CM, Valckx D, Debrock S, Mwenda JM, et al. (2005)
Validation of real-time RT-PCR assays for mRNA quantification in baboons.
Cytokine 31: 454–458.
99. Koga T, Lim JH, Jono H, Ha UH, Xu H, et al. (2008) Tumor suppressor
cylindromatosis acts as a negative regulator for Streptococcus pneumoniae-
induced NFAT signaling. J Biol Chem 283: 12546–12554.
100. Ferrara G, Bleck B, Richeldi L, Reibman J, Fabbri LM, et al. (2008)
Mycobacterium tuberculosis induces CCL18 expression in human macro-
phages. Scand J Immunol 68: 668–674.
101. Peller S, Tabach Y, Rotschild M, Garach-Joshua O, Cohen Y, et al. (2009)
Identification of gene networks associated with erythroid differentiation. Blood
Cells Mol Dis 43: 74–80.
hBD-3 Regulates TAM via CCR2
PLoS ONE | www.plosone.org 14 June 2010 | Volume 5 | Issue 6 | e10993