Table 1.
Chemotactic molecules involved in inflammatory cell trafficking.
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).
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).
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).
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 mm3, while in hBD-3 overexpressed tumors, the value is 66.9 mm3. *, p<0.05.
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.
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-1α, IL-6, IL-8, CCL18, and TNF-α in THP-1 cell-differentiated macrophages treated with hBD-3 (10 µg/ml) for 16 h. (D) Real-time quantitative RT-PCR of IL-1α, IL-6, IL-8, CCL18, and TNF-α in PBM-differentiated macrophages treated with hBD-3 (10 µg/ml) for 16 h. Experiments were repeated 3 times. p values are presented in each graph.
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 µg/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-1α (control). Cells were pretreated with RS102895 at 20 µM for 2 h, followed by cell migration assays. hBD-3, 200 ng/ml; MCP-1, 30 ng/ml; SDF-1α, 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.
Table 2.
Primers used in real-time quantitative RT-PCR.