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, 6 (24), 20002-25

Bisphosphonate-induced Differential Modulation of Immune Cell Function in Gingiva and Bone Marrow in Vivo: Role in Osteoclast-Mediated NK Cell Activation

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Bisphosphonate-induced Differential Modulation of Immune Cell Function in Gingiva and Bone Marrow in Vivo: Role in Osteoclast-Mediated NK Cell Activation

Han-Ching Tseng et al. Oncotarget.

Abstract

The aim of this study is to establish osteoclasts as key immune effectors capable of activating the function of Natural Killer (NK) cells, and expanding their numbers, and to determine in vivo and in vitro effect of bisphosphonates (BPs) during NK cell interaction with osteoclasts and on systemic and local immune function. The profiles of 27 cytokines, chemokines and growth factors released from osteoclasts were found to be different from dendritic cells and M1 macrophages but resembling to untreated monocytes and M2 macrophages. Nitrogen-containing BPs Zoledronate (ZOL) and Alendronate (ALN), but not non-nitrogen-containing BPs Etidronate (ETI), triggered increased release of pro-inflammatory mediators from osteoclasts while all three BPs decreased pit formation by osteoclasts. ZOL and ALN mediated significant release of IL-6, TNF-` and IL-1β, whereas they inhibited IL-10 secretion by osteoclasts. Treatment of osteoclasts with ZOL inhibited NK cell mediated cytotoxicity whereas it induced significant secretion of cytokines and chemokines. NK cells lysed osteoclasts much more than their precursor cells monocytes, and this correlated with the decreased expression of MHC class I expression on osteoclasts. Intravenous injection of ZOL in mice induced pro-inflammatory microenvironment in bone marrow and demonstrated significant immune activation. By contrast, tooth extraction wound of gingival tissues exhibited profound immune suppressive microenvironment associated with dysregulated wound healing to the effect of ZOL which could potentially be responsible for the pathogenesis of Osteonecrosis of the Jaw (ONJ). Finally, based on the data obtained in this paper we demonstrate that osteoclasts can be used as targets for the expansion of NK cells with superior function for immunotherapy of cancer.

Keywords: Immune response; Immunity; Immunology and Microbiology Section; NK cell; bisphosphonate; etidronate; osteoclasts; zoledronate.

Figures

Figure 1
Figure 1. Osteoclasts are susceptible to NK cells mediated cytotoxicity and induce significant levels of IFN-γ secretion by the NK cells
A. Highly purified NK cells (1 × 106 cells/ml) were left untreated, treated with IL-2 (1000 units/ml) or a combination of IL-2 (1000 units/ml) and anti-CD16mAb (3 μg/ml) for 18 hours before they were added to 51Cr labeled autologous osteoclasts at various effector to target ratios. NK cell mediated cytotoxicities were determined using a standard 4 hour 51Cr release assay. B. NK cells were purified and treated with IL-2 (1000 units/ml) for 18 hours and then added to 51Cr labeled autologous monocytes or autologous osteoclasts (hOC) at various effector to target ratios. NK cell mediated cytotoxicities were determined using a standard 4 hour 51Cr release assay. * The difference between IL-2 treated NK cells cultured with monocytes or hOC is significant at p < 0.05. The lytic units 30/106 cells were determined using inverse number of NK cells required to lyse 30% of the target cells X100. C. DCs, hOC and monocytes were prepared as described in Materials and Methods and seeded at 4 × 105 cells/well in 12 well plates and incubated overnight. Purified NK cells were pre-treated with IL-2 (1000 units/mL) or a combination of IL-2 (1000 units/mL) and anti-CD16mAb hOCs for 18 hours and then cultured alone or with autologous DCs, hOC or monocytes at an effector to target ratio of 1:1. The supernatants from each culture condition were harvested overnight, day 3 and day 6. The level of IFN-γ produced by NK cells was measured using multiplex cytokine array kit. D. On the last day of the experimental period, the culture medium was refreshed and cells were cultured for an additional 5 hours. The level of IFN-γ produced by NK cells was measured with a specific ELISA. * The difference between IL-2 or IL-2+anti-CD16mAb treated NK cells cultured with DCs or monocytes compared to IL-2 or IL-2+anti-CD16mAb treated NK cells cultured with hOC is significant at p < 0.05 E. Monocytes, DCs and hOC were co-cultured with pre-treated NK cells, as described in Fig. 1C, and on the last day of the experimental period the number of cells was assessed by microscopic evaluation F. Pre-activated NK cells, as described in Fig. 1A, were co-cultured with autologous DCs, osteoclasts or monocytes for 6 days. Afterwards, the NK cells were divided into two groups-Untreated or treated with additional IL-2 (1000 units/ml) for 48 hours and used in a standard 4 hour 51Cr release assay against OSCSCs. The lytic units 30/106 cells were determined using inverse number of NK cells required to lyse 30% of OSCSCs X100.
Figure 1
Figure 1. Osteoclasts are susceptible to NK cells mediated cytotoxicity and induce significant levels of IFN-γ secretion by the NK cells
A. Highly purified NK cells (1 × 106 cells/ml) were left untreated, treated with IL-2 (1000 units/ml) or a combination of IL-2 (1000 units/ml) and anti-CD16mAb (3 μg/ml) for 18 hours before they were added to 51Cr labeled autologous osteoclasts at various effector to target ratios. NK cell mediated cytotoxicities were determined using a standard 4 hour 51Cr release assay. B. NK cells were purified and treated with IL-2 (1000 units/ml) for 18 hours and then added to 51Cr labeled autologous monocytes or autologous osteoclasts (hOC) at various effector to target ratios. NK cell mediated cytotoxicities were determined using a standard 4 hour 51Cr release assay. * The difference between IL-2 treated NK cells cultured with monocytes or hOC is significant at p < 0.05. The lytic units 30/106 cells were determined using inverse number of NK cells required to lyse 30% of the target cells X100. C. DCs, hOC and monocytes were prepared as described in Materials and Methods and seeded at 4 × 105 cells/well in 12 well plates and incubated overnight. Purified NK cells were pre-treated with IL-2 (1000 units/mL) or a combination of IL-2 (1000 units/mL) and anti-CD16mAb hOCs for 18 hours and then cultured alone or with autologous DCs, hOC or monocytes at an effector to target ratio of 1:1. The supernatants from each culture condition were harvested overnight, day 3 and day 6. The level of IFN-γ produced by NK cells was measured using multiplex cytokine array kit. D. On the last day of the experimental period, the culture medium was refreshed and cells were cultured for an additional 5 hours. The level of IFN-γ produced by NK cells was measured with a specific ELISA. * The difference between IL-2 or IL-2+anti-CD16mAb treated NK cells cultured with DCs or monocytes compared to IL-2 or IL-2+anti-CD16mAb treated NK cells cultured with hOC is significant at p < 0.05 E. Monocytes, DCs and hOC were co-cultured with pre-treated NK cells, as described in Fig. 1C, and on the last day of the experimental period the number of cells was assessed by microscopic evaluation F. Pre-activated NK cells, as described in Fig. 1A, were co-cultured with autologous DCs, osteoclasts or monocytes for 6 days. Afterwards, the NK cells were divided into two groups-Untreated or treated with additional IL-2 (1000 units/ml) for 48 hours and used in a standard 4 hour 51Cr release assay against OSCSCs. The lytic units 30/106 cells were determined using inverse number of NK cells required to lyse 30% of OSCSCs X100.
Figure 2
Figure 2. Uptake and the pro-inflammatory effect of ZOL and ALN on osteoclasts
A. Human osteoclasts were generated from healthy donor's monocytes as described in the Materials and Methods section. Afterwards, osteoclasts were washed with 1X PBS, detached from the tissue culture plate and seeded at a density of 3 × 104 cells/well in 24 well plate. After an overnight incubation, the cells were treated with ZOL conjugated with 5 - Carboxy - X - rhodamine (5 μM) for 24 hours and the image was taken with Leica DMI 6000B microscope B. Osteoclasts were prepared as described in the Materials and Methods section and seeded at 1 × 104 cells/well in 24 well plates. The osteoclasts were then treated with ZOL or ALN (1 μM) for 7 days. Afterwards, culture medium was removed and the cells were washed with 1X PBS and fixed with solution containing citrate solution, acetone and 37% formaldehyde for 5 minutes. The cells were then washed with water and photographed using Leica DMI 6000B inverted microscope C. Osteoclasts were prepared as described in the Materials and Methods section and seeded at 1 × 104 cells/well in 24 well plates. The osteoclasts were then treated with ZOL or ALN (1 μM) for 7 days. Afterwards, culture medium was removed and the cells were rinsed with 1X PBS and fixed with solution containing citrate solution, acetone and 37% formaldehyde for 5 minutes. Cells were washed with twice with 1XPBS before Fast Garnet GBC and sodium nitrite at (1:1 ratio) were added and incubated for 1 hour at 37° (C) Plates were rinsed and hematoxylin were then added to each well for 2 mins. Plates were air dried and images were taken by the Leica DMI 6000B inverted microscope.
Figure 3
Figure 3. Decreased size, pit numbers, viability and cytokine secretion mediated by ZOL, ALN, and ETI-treated osteoclasts
A. Osteoclasts were generated as described in the Materials and Methods section for 12 days. Osteoclasts (2.5 × 105 cells/ml) were treated with different concentrations (10 nM-50 μM) of ZOL for 48 hours, and afterwards the cells were washed using freshly prepared 5% sodium hypochlorite and the images were taken with Leica DMI 6000B inverted microscope at 5X magnification B. Osteoclasts were prepared as described in Fig. 3A and treated with varying concentrations (10 nM-50 μM) of ZOL, ALN or ETI for 48 hours. Afterwards, the number of pits was counted using microscopy at 5X magnification C. Purified osteoclasts were treated with varying concentrations of ZOL, ALN and ETI for 48 hours, afterwards the cells were washed with 1X PBS, stained with propidium iodide and analyzed by flow cytometry D. Osteoclasts were generated as described in the Materials and Methods section. After the differentiation period, osteoclasts were washed with 1X PBS, detached from tissue culture plates and seeded at 1.5 × 104 cells/well in 24 well plate for 24 hours. Cells were then left untreated or treated with ZOL, ALN or ETI at 1, 5, 10, 25, 50 or 100 μM for 6 days. Supernatants were removed on the last day of experiment and the levels of IL-6 E. and IL-10 F. were determined with specific ELISAs. * The difference between osteoclasts treated with ZOL or ALN compared to untreated osteoclasts is significant at P < 0.05. Osteoclasts were generated as described in the Materials and Methods section for 12 days. Afterwards, osteoclasts were seeded at 2 × 105 cells/ml in 24 well plate for 24 hours, and treated with ZOL, ALN or ETI at 10 nM, 100 nM, 500 nM or 1 μM for 3 days after which the supernatants were harvested and the levels of IL-6 G. IL-10 H. and TNF-α (I). were determined with specific ELISAs. * The difference between osteoclasts treated with ZOL or ALN compared to untreated osteoclasts is significant at P < 0.05.
Figure 4
Figure 4. Osteoclasts treated with supernatants from activated NKs or ZOL demonstrated modulation of cell surface receptors
A. Osteoclasts were generated as described in the Materials and Methods section for 17 days. Osteoclasts were then left untreated or treated with ZOL at 500 nM or 5 μM. After 4 days of treatment, surface expression of CD54, MHC-I, MHC-II, CD44, CD14, CD11b and B7H1 were determined using flow cytometric analysis after staining with the respective PE-conjugated antibodies B. Osteoclasts were left untreated or treated with ZOL (1 μM) or supernatants harvested from pre-activated NK cells cultured with monocytes and probiotic bacteria (sAJ2) for 48 hours. Surface expressions of CD54, MHC-I, MHC-II, CD44, CD14 and CD11b were determined using flow cytometric analysis after staining with the respective PE-conjugated antibodies C. Isotype control antibodies were used as control. The numbers on the right hand corner are the percentages and the mean channel fluoresence intensities for each histogram.
Figure 5
Figure 5. ZOL-treated osteoclasts and OSCSCs were resistant to NK-mediated cytotoxicity and secreted high levels of IFN-γ
A. Highly purified NK cells (1 × 106 cells/ml) were left untreated or treated with IL-2 (1000 units/ml) or a combination of IL-2 (1000 units/ml) and anti-CD16mAb (3 μg/ml) for 18 hours before they were added to 51Cr labeled osteoclasts (hOC) at various effector to target ratios. Osteoclasts were prepared as described in the Materials and Methods section and treated with ZOL (250 nM, 500 nM or 1 uM) for 30 minutes before they were used as target cells. NK cell mediated cytotoxicity was determined using a standard 4 hour 51Cr release assay. * The difference between IL-2 or IL-2+anti-CD16mAb stimulated NK cells treated with ZOL compared to IL-2 or IL-2+anti-CD16mAb stimulated NK cells without ZOL treatment is significant at P < 0.05 B. Osteoclasts were prepared as described in the Materials and Methods section and treated with ZOL, ALN or ETI (100 nM) for 30 minutes before used as target cells. NK cells were prepared as described in Fig. 5A and then added to 51Cr labeled osteoclasts at various effector to target ratios. NK cell mediated cytotoxicity was determined using a standard 4 hour 51Cr release assay. * The difference between IL-2 or IL-2+anti-CD16mAb stimulated NK cells treated with ZOL or ALN compared to IL-2 or IL-2+anti-CD16mAb stimulated NK cells without BP treatment is significant at P < 0.05 C. OSCSCs were treated with ZOL, ALN or ETI (1 μM) for 30 minutes before the addition of pre-treated NK cells, prepared as described in Fig. 5A. NK cell mediated cytotoxicity was determined using a standard 4 hour 51Cr release assay. * The difference between IL-2 stimulated NK cells treated with ZOL compared to IL-2 treated NK cells without BP treatment is significant at P < 0.05 D. Osteoclasts were differentiated from autologous monocytes as described in the Materials and Methods section for 17 days. NK cells were left untreated or treated with IL-2 (1000 units/ml) in the presence and absence of ZOL (500 nM) and ALN (500 nM) for 18 hours before they were added to 51Cr labeled osteoclasts at various effector to target ratios. NK cell mediated cytotoxicity was determined using a standard 4 hour 51Cr release assay. * The difference between IL-2 stimulated NK cells treated with ZOL or ALN compared to IL-2 stimulated NK cells without BP treatment is significant at P < 0.05. The lytic units 30/106 cells were determined using inverse number of NK cells required to lyse 30% of the osteoclasts or OSCSCs X100. E. Osteoclasts were treated as described in Fig. 5B and then added to untreated, IL-2 treated or IL-2+anti-CD16mAb treated NK cells at 1:3 ratio (NK: target cells). After an overnight incubation, the supernatants were harvested and the levels of IL-6 F. IFN-γ G. and IL-18 were measured with specific ELISAs. * The difference between IL-2 or IL-2+anti-CD16mAb stimulated NK cells treated with ZOL or ALN compared to IL-2 or IL-2+anti-CD16mAb stimulated NK cells without BP treatment is significant at P < 0.05 (E) * The difference between IL-2 stimulated NK cells treated with ZOL compared to IL-2 stimulated NK cells without BP treatment is significant at P < 0.05 (F) * The difference between untreated, IL-2 or IL-2+anti-CD16mAb stimulated NK cells treated with ZOL compared to those NK cells without BP treatment is significant at P < 0.05.
Figure 6
Figure 6. ZOL-treated murine osteoclasts trigger significant secretion of IFN-γ by NK, CD3+ T cells and γδ T cells
A. Monocytes were purified from bone marrow cells extracted from femurs of WT B6 mice. Osteoclasts were generated as described in the Materials and Methods section, treated with or without ZOL (500 nM) for 12 hours and then washed extensively before they were cultured with pre-activated NK cells (1 × 106 cells/ml) treated with IL-2 (10, 000 units/ml for 12 hours) at 2:1 ratio (NK cells: osteoclasts). At the time of the co-culture of NK cells with osteoclasts the cultures were supplemented with LPS (100 ng/ml) and incubated for 48 hours before they were washed and treated with only IL-2 (10, 000 units/ml) for an additional 48 hours, after which the supernatants were harvested and the levels of secreted IFN-γ were determined using specific ELISAs. * The difference between IL-2 treated NK cells cultured with untreated OCs or ZOL treated OCs compared to IL-2 treated NK cells cultured in media is significant at P < 0.05 B. Osteoclasts, and highly purified T cells were prepared and co-cultured as described for NK cells and co-cultured as described in Fig. 6A. * The difference between IL-2 treated T cells cultured with untreated OCs or ZOL treated OCs compared to IL-2 treated T cells cultured in media is significant at P < 0.05 C. Osteoclasts, and highly purified γδT cells were prepared and co-cultured as described for NK cells and co-cultured as described in Fig. 6A. * The difference between IL-2 treated γδT cells cultured with untreated OCs or ZOL treated OCs compared to IL-2 treated γδT cells cultured in media is significant at P < 0.05 (C) After 4 days of culture, the supernatants were harvested and the levels of IFN-γ were measured with specific ELISAs.
Figure 7
Figure 7. In vivo injection of ZOL triggered significant IFN-γ and IL-6 secretion by bone marrow-derived cells but inhibited secretion by gingival cells
A. Female Balb/c mice received IV injection of 500 μg/Kg ZOL or 0.9% NACL vehicle solution followed by maxillary first molar extraction after 6 days. Two weeks after tooth extraction, the maxillary palatal mucosa of 0.9% NACL solution-injected control mice showed complete wound healing, whereas tissue swelling (dotted line) around the tooth extraction site remained in ZOL-injected mice. The maxillary bone of FAM-ZOL-injected mice showed the strong fluorescence signal, while the remaining molars (M1, M2, M3) were absent of fluorescence. The visibly reduced fluorescence was observed in the tooth extraction sockets (*) and the adjacent palatal bone area (dotted line and arrowheads) B. Hematoxylin and eosin (H & E) stained histological sections of ZOL-injected mice showed significant inflammatory cell infiltration (Inflam) in the palatal/gingival soft tissue one week after tooth extraction. There were signs of abnormal epithelial hyperplasia (Epi.Hyp.: arrows). The extraction socket (*) exhibited new bone formation. TRAP staining revealed a number of osteoclasts (vertical arrows) on the surface of palatal bone (Palat.Bone) as well as in the extraction socket (*). TRAP+ multinuclear cells were found within the inflammation area (arrowheads). In non-decalcified cross-section of FAM-ZOL-injected mice, FAM-positive large cells (arrows) were found on the palatal bone as well as away from the bone surface (arrowheads). Taken together, these cells in the inflammatory area were thought to be ZOL-affected osteoclasts C. Immunohistology for cytokeratin 14 (CK14) was performed as described in the Materials and Method section, and confirmed oral epithelial hyperplasia in the palatal/gingival tissue of ZOL-injected mice (Epi.Hyp.: arrows). Epithelial hyperplasia was primarily observed within the strong inflammatory area D. Female B6 mice received IV injection of 500 μg/Kg ZOL or 0.9% NACL vehicle solution followed by maxillary first molar extraction after 6 days. Two weeks after tooth extraction the surface expression of CD45FITC+CD3PE or CD45FITC+DX5PE on cells obtained from gingival tissues. E. and bone marrow F. were assessed with flow cytometric analysis after staining with the respective PE- and FITC-conjugated antibodies. Isotype control antibodies were used as control. Right upper quadrant represent the percentage of CD3 and DX5 positive cells within the CD45 population. B6 mice underwent experimental treatment as described in Fig. 7D. Gingival tissues obtained from ZOL and NACL injected animals were digested as described in Materials and Method. Dissociated cells obtained from gingival tissues were cultured in the presence of IL-2 (10,000 units/ml). Supernatants were harvested 5 days after IL-2 treatment. The mean for (n = 4) is shown for each set in the figure. The difference between ZOL and NACL injected gingivae is significant at a P < 0.05 G. After euthanasia, femurs from ZOL and NACL injected animals were harvested and bone marrow cells were extracted and cultured (1 × 106 /ml) in the presence of IL-2 (10, 000 units/ml). Supernatants were harvested 5 days after IL-2 treatment. The mean for (n = 4) is shown for each set in the figure. The difference between ZOL and NACL injected bone marrow cells is significant at a P < 0.05 H. Gingival tissue, pancreas and adipose (1 × 106 /ml) from ZOL and NACL injected animals were cultured in the absence or presence of IL-2 (10, 000 units/ml) for 5 days, after which the supernatants were harvested and the levels of IFN-γ were measured with specific ELISA. *The difference between untreated or IL-2 stimulated cells from ZOL injected mice compared to untreated or IL-2 stimulated cells from NACL injected mice is significant at P < 0.05 I. Female WT B6 mice received IV injection of ZOL or NACL as described in Fig. 7A. After euthanasia, gingival tissues obtained from ZOL and NACL injected mice were digested as described in Materials and Methods section and cultured (1 × 106 /ml) in the presence of IL-2 (10, 000 units/ml). After five days of culture, supernatants from the cultures of gingival tissue were harvested and the levels of IL-6 secretion were measured by specific ELISA. J. Immunohistology for IL-6 was performed as described in the Materials and Methods section, and showed the IL-6 immuno-localization pattern was distinctly was different in osteoclasts in the jawbone of 0.9% NACL solution-injected control mice and ZOL-injected mice K. Female B6 mice received IV injection of ZOL or NACL as described in Fig. 7A. After euthanasia, femurs from ZOL and NACL injected mice were harvested and bone marrow cells were extracted and cultured (1 × 106 /ml) in the presence and absence of IL-2 (10,000 units/ml). After five days of culture, the supernatants were removed and the levels of IL-6 secretion were measured by specific ELISA. L. The pancreas was obtained from ZOL and NACL injected mice and digested as described in Materials and Methods section. Dissociated pancreatic cells were cultured in the presence and absence of IL-2 (10, 000 units/ml). After five days of culture, the supernatants were removed and the levels of IL-6 secretion were measured by specific ELISA M. Dissociated gingival, pancreas and adipose cells (1 × 106 /ml) from ZOL and NACL injected animals were cultured in the absence or presence of IL-2 (10, 000 units/ml) for 5 days and used as effectors against 51Cr labeled ST63 cells at various effector to target ratios in a standard 4 hour 51Cr release assay. The lytic units 30/106 cells were determined using inverse number of NK cells required to lyse 30% of the ST63 cells X100 N. Total cells from the bone marrow were prepared as described in Fig. 7K O. were prepared as described in Fig. 7K. NK cells were purified from bone marrow cells and cultured in the presence or absence of IL-2 (10, 000 units/ml) for 5 days P. Afterwards, the cells were used as effectors against 51Cr labeled ST63 cells at various effector to target ratios in a standard 4 hour 51Cr release assay. The lytic units 30/106 cells were determined using inverse number of NK cells required to lyse 30% of ST63 cells X100. * The difference between IL-2 treated BM (N) or BM-NK (O) cells obtained from mice injected with ZOL compared to IL-2 treated BM or BM-NK cells from mice injected with NACL is significant at P < 0.05. NK cells purified from bone marrow were cultured in the presence and absence of IL-2 (10, 000 units/ml) after which supernatants were harvested at day 5 and the levels of IFN-γ Q. and IL-6 R. were determined by specific ELISAs. One week after tooth extraction, total RNA samples were isolated from palatal/gingival tissue of control and ZOL-injected mice. PCR microarray analysis revealed the suppressed expression of pro-inflammatory cytokines such as IFN-γ (ifng), IL-6, -12b, -15, -2 and TNF-α (Tnf) in the ZOL-injected mice. Those unaffected cytokines included anti-inflammatory cytokines such as IL-10 and -4 (R) (A complete list of PCR microarray data can be found in Fig. S3).
Figure 7
Figure 7. In vivo injection of ZOL triggered significant IFN-γ and IL-6 secretion by bone marrow-derived cells but inhibited secretion by gingival cells
A. Female Balb/c mice received IV injection of 500 μg/Kg ZOL or 0.9% NACL vehicle solution followed by maxillary first molar extraction after 6 days. Two weeks after tooth extraction, the maxillary palatal mucosa of 0.9% NACL solution-injected control mice showed complete wound healing, whereas tissue swelling (dotted line) around the tooth extraction site remained in ZOL-injected mice. The maxillary bone of FAM-ZOL-injected mice showed the strong fluorescence signal, while the remaining molars (M1, M2, M3) were absent of fluorescence. The visibly reduced fluorescence was observed in the tooth extraction sockets (*) and the adjacent palatal bone area (dotted line and arrowheads) B. Hematoxylin and eosin (H & E) stained histological sections of ZOL-injected mice showed significant inflammatory cell infiltration (Inflam) in the palatal/gingival soft tissue one week after tooth extraction. There were signs of abnormal epithelial hyperplasia (Epi.Hyp.: arrows). The extraction socket (*) exhibited new bone formation. TRAP staining revealed a number of osteoclasts (vertical arrows) on the surface of palatal bone (Palat.Bone) as well as in the extraction socket (*). TRAP+ multinuclear cells were found within the inflammation area (arrowheads). In non-decalcified cross-section of FAM-ZOL-injected mice, FAM-positive large cells (arrows) were found on the palatal bone as well as away from the bone surface (arrowheads). Taken together, these cells in the inflammatory area were thought to be ZOL-affected osteoclasts C. Immunohistology for cytokeratin 14 (CK14) was performed as described in the Materials and Method section, and confirmed oral epithelial hyperplasia in the palatal/gingival tissue of ZOL-injected mice (Epi.Hyp.: arrows). Epithelial hyperplasia was primarily observed within the strong inflammatory area D. Female B6 mice received IV injection of 500 μg/Kg ZOL or 0.9% NACL vehicle solution followed by maxillary first molar extraction after 6 days. Two weeks after tooth extraction the surface expression of CD45FITC+CD3PE or CD45FITC+DX5PE on cells obtained from gingival tissues. E. and bone marrow F. were assessed with flow cytometric analysis after staining with the respective PE- and FITC-conjugated antibodies. Isotype control antibodies were used as control. Right upper quadrant represent the percentage of CD3 and DX5 positive cells within the CD45 population. B6 mice underwent experimental treatment as described in Fig. 7D. Gingival tissues obtained from ZOL and NACL injected animals were digested as described in Materials and Method. Dissociated cells obtained from gingival tissues were cultured in the presence of IL-2 (10,000 units/ml). Supernatants were harvested 5 days after IL-2 treatment. The mean for (n = 4) is shown for each set in the figure. The difference between ZOL and NACL injected gingivae is significant at a P < 0.05 G. After euthanasia, femurs from ZOL and NACL injected animals were harvested and bone marrow cells were extracted and cultured (1 × 106 /ml) in the presence of IL-2 (10, 000 units/ml). Supernatants were harvested 5 days after IL-2 treatment. The mean for (n = 4) is shown for each set in the figure. The difference between ZOL and NACL injected bone marrow cells is significant at a P < 0.05 H. Gingival tissue, pancreas and adipose (1 × 106 /ml) from ZOL and NACL injected animals were cultured in the absence or presence of IL-2 (10, 000 units/ml) for 5 days, after which the supernatants were harvested and the levels of IFN-γ were measured with specific ELISA. *The difference between untreated or IL-2 stimulated cells from ZOL injected mice compared to untreated or IL-2 stimulated cells from NACL injected mice is significant at P < 0.05 I. Female WT B6 mice received IV injection of ZOL or NACL as described in Fig. 7A. After euthanasia, gingival tissues obtained from ZOL and NACL injected mice were digested as described in Materials and Methods section and cultured (1 × 106 /ml) in the presence of IL-2 (10, 000 units/ml). After five days of culture, supernatants from the cultures of gingival tissue were harvested and the levels of IL-6 secretion were measured by specific ELISA. J. Immunohistology for IL-6 was performed as described in the Materials and Methods section, and showed the IL-6 immuno-localization pattern was distinctly was different in osteoclasts in the jawbone of 0.9% NACL solution-injected control mice and ZOL-injected mice K. Female B6 mice received IV injection of ZOL or NACL as described in Fig. 7A. After euthanasia, femurs from ZOL and NACL injected mice were harvested and bone marrow cells were extracted and cultured (1 × 106 /ml) in the presence and absence of IL-2 (10,000 units/ml). After five days of culture, the supernatants were removed and the levels of IL-6 secretion were measured by specific ELISA. L. The pancreas was obtained from ZOL and NACL injected mice and digested as described in Materials and Methods section. Dissociated pancreatic cells were cultured in the presence and absence of IL-2 (10, 000 units/ml). After five days of culture, the supernatants were removed and the levels of IL-6 secretion were measured by specific ELISA M. Dissociated gingival, pancreas and adipose cells (1 × 106 /ml) from ZOL and NACL injected animals were cultured in the absence or presence of IL-2 (10, 000 units/ml) for 5 days and used as effectors against 51Cr labeled ST63 cells at various effector to target ratios in a standard 4 hour 51Cr release assay. The lytic units 30/106 cells were determined using inverse number of NK cells required to lyse 30% of the ST63 cells X100 N. Total cells from the bone marrow were prepared as described in Fig. 7K O. were prepared as described in Fig. 7K. NK cells were purified from bone marrow cells and cultured in the presence or absence of IL-2 (10, 000 units/ml) for 5 days P. Afterwards, the cells were used as effectors against 51Cr labeled ST63 cells at various effector to target ratios in a standard 4 hour 51Cr release assay. The lytic units 30/106 cells were determined using inverse number of NK cells required to lyse 30% of ST63 cells X100. * The difference between IL-2 treated BM (N) or BM-NK (O) cells obtained from mice injected with ZOL compared to IL-2 treated BM or BM-NK cells from mice injected with NACL is significant at P < 0.05. NK cells purified from bone marrow were cultured in the presence and absence of IL-2 (10, 000 units/ml) after which supernatants were harvested at day 5 and the levels of IFN-γ Q. and IL-6 R. were determined by specific ELISAs. One week after tooth extraction, total RNA samples were isolated from palatal/gingival tissue of control and ZOL-injected mice. PCR microarray analysis revealed the suppressed expression of pro-inflammatory cytokines such as IFN-γ (ifng), IL-6, -12b, -15, -2 and TNF-α (Tnf) in the ZOL-injected mice. Those unaffected cytokines included anti-inflammatory cytokines such as IL-10 and -4 (R) (A complete list of PCR microarray data can be found in Fig. S3).
Figure 7
Figure 7. In vivo injection of ZOL triggered significant IFN-γ and IL-6 secretion by bone marrow-derived cells but inhibited secretion by gingival cells
A. Female Balb/c mice received IV injection of 500 μg/Kg ZOL or 0.9% NACL vehicle solution followed by maxillary first molar extraction after 6 days. Two weeks after tooth extraction, the maxillary palatal mucosa of 0.9% NACL solution-injected control mice showed complete wound healing, whereas tissue swelling (dotted line) around the tooth extraction site remained in ZOL-injected mice. The maxillary bone of FAM-ZOL-injected mice showed the strong fluorescence signal, while the remaining molars (M1, M2, M3) were absent of fluorescence. The visibly reduced fluorescence was observed in the tooth extraction sockets (*) and the adjacent palatal bone area (dotted line and arrowheads) B. Hematoxylin and eosin (H & E) stained histological sections of ZOL-injected mice showed significant inflammatory cell infiltration (Inflam) in the palatal/gingival soft tissue one week after tooth extraction. There were signs of abnormal epithelial hyperplasia (Epi.Hyp.: arrows). The extraction socket (*) exhibited new bone formation. TRAP staining revealed a number of osteoclasts (vertical arrows) on the surface of palatal bone (Palat.Bone) as well as in the extraction socket (*). TRAP+ multinuclear cells were found within the inflammation area (arrowheads). In non-decalcified cross-section of FAM-ZOL-injected mice, FAM-positive large cells (arrows) were found on the palatal bone as well as away from the bone surface (arrowheads). Taken together, these cells in the inflammatory area were thought to be ZOL-affected osteoclasts C. Immunohistology for cytokeratin 14 (CK14) was performed as described in the Materials and Method section, and confirmed oral epithelial hyperplasia in the palatal/gingival tissue of ZOL-injected mice (Epi.Hyp.: arrows). Epithelial hyperplasia was primarily observed within the strong inflammatory area D. Female B6 mice received IV injection of 500 μg/Kg ZOL or 0.9% NACL vehicle solution followed by maxillary first molar extraction after 6 days. Two weeks after tooth extraction the surface expression of CD45FITC+CD3PE or CD45FITC+DX5PE on cells obtained from gingival tissues. E. and bone marrow F. were assessed with flow cytometric analysis after staining with the respective PE- and FITC-conjugated antibodies. Isotype control antibodies were used as control. Right upper quadrant represent the percentage of CD3 and DX5 positive cells within the CD45 population. B6 mice underwent experimental treatment as described in Fig. 7D. Gingival tissues obtained from ZOL and NACL injected animals were digested as described in Materials and Method. Dissociated cells obtained from gingival tissues were cultured in the presence of IL-2 (10,000 units/ml). Supernatants were harvested 5 days after IL-2 treatment. The mean for (n = 4) is shown for each set in the figure. The difference between ZOL and NACL injected gingivae is significant at a P < 0.05 G. After euthanasia, femurs from ZOL and NACL injected animals were harvested and bone marrow cells were extracted and cultured (1 × 106 /ml) in the presence of IL-2 (10, 000 units/ml). Supernatants were harvested 5 days after IL-2 treatment. The mean for (n = 4) is shown for each set in the figure. The difference between ZOL and NACL injected bone marrow cells is significant at a P < 0.05 H. Gingival tissue, pancreas and adipose (1 × 106 /ml) from ZOL and NACL injected animals were cultured in the absence or presence of IL-2 (10, 000 units/ml) for 5 days, after which the supernatants were harvested and the levels of IFN-γ were measured with specific ELISA. *The difference between untreated or IL-2 stimulated cells from ZOL injected mice compared to untreated or IL-2 stimulated cells from NACL injected mice is significant at P < 0.05 I. Female WT B6 mice received IV injection of ZOL or NACL as described in Fig. 7A. After euthanasia, gingival tissues obtained from ZOL and NACL injected mice were digested as described in Materials and Methods section and cultured (1 × 106 /ml) in the presence of IL-2 (10, 000 units/ml). After five days of culture, supernatants from the cultures of gingival tissue were harvested and the levels of IL-6 secretion were measured by specific ELISA. J. Immunohistology for IL-6 was performed as described in the Materials and Methods section, and showed the IL-6 immuno-localization pattern was distinctly was different in osteoclasts in the jawbone of 0.9% NACL solution-injected control mice and ZOL-injected mice K. Female B6 mice received IV injection of ZOL or NACL as described in Fig. 7A. After euthanasia, femurs from ZOL and NACL injected mice were harvested and bone marrow cells were extracted and cultured (1 × 106 /ml) in the presence and absence of IL-2 (10,000 units/ml). After five days of culture, the supernatants were removed and the levels of IL-6 secretion were measured by specific ELISA. L. The pancreas was obtained from ZOL and NACL injected mice and digested as described in Materials and Methods section. Dissociated pancreatic cells were cultured in the presence and absence of IL-2 (10, 000 units/ml). After five days of culture, the supernatants were removed and the levels of IL-6 secretion were measured by specific ELISA M. Dissociated gingival, pancreas and adipose cells (1 × 106 /ml) from ZOL and NACL injected animals were cultured in the absence or presence of IL-2 (10, 000 units/ml) for 5 days and used as effectors against 51Cr labeled ST63 cells at various effector to target ratios in a standard 4 hour 51Cr release assay. The lytic units 30/106 cells were determined using inverse number of NK cells required to lyse 30% of the ST63 cells X100 N. Total cells from the bone marrow were prepared as described in Fig. 7K O. were prepared as described in Fig. 7K. NK cells were purified from bone marrow cells and cultured in the presence or absence of IL-2 (10, 000 units/ml) for 5 days P. Afterwards, the cells were used as effectors against 51Cr labeled ST63 cells at various effector to target ratios in a standard 4 hour 51Cr release assay. The lytic units 30/106 cells were determined using inverse number of NK cells required to lyse 30% of ST63 cells X100. * The difference between IL-2 treated BM (N) or BM-NK (O) cells obtained from mice injected with ZOL compared to IL-2 treated BM or BM-NK cells from mice injected with NACL is significant at P < 0.05. NK cells purified from bone marrow were cultured in the presence and absence of IL-2 (10, 000 units/ml) after which supernatants were harvested at day 5 and the levels of IFN-γ Q. and IL-6 R. were determined by specific ELISAs. One week after tooth extraction, total RNA samples were isolated from palatal/gingival tissue of control and ZOL-injected mice. PCR microarray analysis revealed the suppressed expression of pro-inflammatory cytokines such as IFN-γ (ifng), IL-6, -12b, -15, -2 and TNF-α (Tnf) in the ZOL-injected mice. Those unaffected cytokines included anti-inflammatory cytokines such as IL-10 and -4 (R) (A complete list of PCR microarray data can be found in Fig. S3).
Figure 7
Figure 7. In vivo injection of ZOL triggered significant IFN-γ and IL-6 secretion by bone marrow-derived cells but inhibited secretion by gingival cells
A. Female Balb/c mice received IV injection of 500 μg/Kg ZOL or 0.9% NACL vehicle solution followed by maxillary first molar extraction after 6 days. Two weeks after tooth extraction, the maxillary palatal mucosa of 0.9% NACL solution-injected control mice showed complete wound healing, whereas tissue swelling (dotted line) around the tooth extraction site remained in ZOL-injected mice. The maxillary bone of FAM-ZOL-injected mice showed the strong fluorescence signal, while the remaining molars (M1, M2, M3) were absent of fluorescence. The visibly reduced fluorescence was observed in the tooth extraction sockets (*) and the adjacent palatal bone area (dotted line and arrowheads) B. Hematoxylin and eosin (H & E) stained histological sections of ZOL-injected mice showed significant inflammatory cell infiltration (Inflam) in the palatal/gingival soft tissue one week after tooth extraction. There were signs of abnormal epithelial hyperplasia (Epi.Hyp.: arrows). The extraction socket (*) exhibited new bone formation. TRAP staining revealed a number of osteoclasts (vertical arrows) on the surface of palatal bone (Palat.Bone) as well as in the extraction socket (*). TRAP+ multinuclear cells were found within the inflammation area (arrowheads). In non-decalcified cross-section of FAM-ZOL-injected mice, FAM-positive large cells (arrows) were found on the palatal bone as well as away from the bone surface (arrowheads). Taken together, these cells in the inflammatory area were thought to be ZOL-affected osteoclasts C. Immunohistology for cytokeratin 14 (CK14) was performed as described in the Materials and Method section, and confirmed oral epithelial hyperplasia in the palatal/gingival tissue of ZOL-injected mice (Epi.Hyp.: arrows). Epithelial hyperplasia was primarily observed within the strong inflammatory area D. Female B6 mice received IV injection of 500 μg/Kg ZOL or 0.9% NACL vehicle solution followed by maxillary first molar extraction after 6 days. Two weeks after tooth extraction the surface expression of CD45FITC+CD3PE or CD45FITC+DX5PE on cells obtained from gingival tissues. E. and bone marrow F. were assessed with flow cytometric analysis after staining with the respective PE- and FITC-conjugated antibodies. Isotype control antibodies were used as control. Right upper quadrant represent the percentage of CD3 and DX5 positive cells within the CD45 population. B6 mice underwent experimental treatment as described in Fig. 7D. Gingival tissues obtained from ZOL and NACL injected animals were digested as described in Materials and Method. Dissociated cells obtained from gingival tissues were cultured in the presence of IL-2 (10,000 units/ml). Supernatants were harvested 5 days after IL-2 treatment. The mean for (n = 4) is shown for each set in the figure. The difference between ZOL and NACL injected gingivae is significant at a P < 0.05 G. After euthanasia, femurs from ZOL and NACL injected animals were harvested and bone marrow cells were extracted and cultured (1 × 106 /ml) in the presence of IL-2 (10, 000 units/ml). Supernatants were harvested 5 days after IL-2 treatment. The mean for (n = 4) is shown for each set in the figure. The difference between ZOL and NACL injected bone marrow cells is significant at a P < 0.05 H. Gingival tissue, pancreas and adipose (1 × 106 /ml) from ZOL and NACL injected animals were cultured in the absence or presence of IL-2 (10, 000 units/ml) for 5 days, after which the supernatants were harvested and the levels of IFN-γ were measured with specific ELISA. *The difference between untreated or IL-2 stimulated cells from ZOL injected mice compared to untreated or IL-2 stimulated cells from NACL injected mice is significant at P < 0.05 I. Female WT B6 mice received IV injection of ZOL or NACL as described in Fig. 7A. After euthanasia, gingival tissues obtained from ZOL and NACL injected mice were digested as described in Materials and Methods section and cultured (1 × 106 /ml) in the presence of IL-2 (10, 000 units/ml). After five days of culture, supernatants from the cultures of gingival tissue were harvested and the levels of IL-6 secretion were measured by specific ELISA. J. Immunohistology for IL-6 was performed as described in the Materials and Methods section, and showed the IL-6 immuno-localization pattern was distinctly was different in osteoclasts in the jawbone of 0.9% NACL solution-injected control mice and ZOL-injected mice K. Female B6 mice received IV injection of ZOL or NACL as described in Fig. 7A. After euthanasia, femurs from ZOL and NACL injected mice were harvested and bone marrow cells were extracted and cultured (1 × 106 /ml) in the presence and absence of IL-2 (10,000 units/ml). After five days of culture, the supernatants were removed and the levels of IL-6 secretion were measured by specific ELISA. L. The pancreas was obtained from ZOL and NACL injected mice and digested as described in Materials and Methods section. Dissociated pancreatic cells were cultured in the presence and absence of IL-2 (10, 000 units/ml). After five days of culture, the supernatants were removed and the levels of IL-6 secretion were measured by specific ELISA M. Dissociated gingival, pancreas and adipose cells (1 × 106 /ml) from ZOL and NACL injected animals were cultured in the absence or presence of IL-2 (10, 000 units/ml) for 5 days and used as effectors against 51Cr labeled ST63 cells at various effector to target ratios in a standard 4 hour 51Cr release assay. The lytic units 30/106 cells were determined using inverse number of NK cells required to lyse 30% of the ST63 cells X100 N. Total cells from the bone marrow were prepared as described in Fig. 7K O. were prepared as described in Fig. 7K. NK cells were purified from bone marrow cells and cultured in the presence or absence of IL-2 (10, 000 units/ml) for 5 days P. Afterwards, the cells were used as effectors against 51Cr labeled ST63 cells at various effector to target ratios in a standard 4 hour 51Cr release assay. The lytic units 30/106 cells were determined using inverse number of NK cells required to lyse 30% of ST63 cells X100. * The difference between IL-2 treated BM (N) or BM-NK (O) cells obtained from mice injected with ZOL compared to IL-2 treated BM or BM-NK cells from mice injected with NACL is significant at P < 0.05. NK cells purified from bone marrow were cultured in the presence and absence of IL-2 (10, 000 units/ml) after which supernatants were harvested at day 5 and the levels of IFN-γ Q. and IL-6 R. were determined by specific ELISAs. One week after tooth extraction, total RNA samples were isolated from palatal/gingival tissue of control and ZOL-injected mice. PCR microarray analysis revealed the suppressed expression of pro-inflammatory cytokines such as IFN-γ (ifng), IL-6, -12b, -15, -2 and TNF-α (Tnf) in the ZOL-injected mice. Those unaffected cytokines included anti-inflammatory cytokines such as IL-10 and -4 (R) (A complete list of PCR microarray data can be found in Fig. S3).
Figure 7
Figure 7. In vivo injection of ZOL triggered significant IFN-γ and IL-6 secretion by bone marrow-derived cells but inhibited secretion by gingival cells
A. Female Balb/c mice received IV injection of 500 μg/Kg ZOL or 0.9% NACL vehicle solution followed by maxillary first molar extraction after 6 days. Two weeks after tooth extraction, the maxillary palatal mucosa of 0.9% NACL solution-injected control mice showed complete wound healing, whereas tissue swelling (dotted line) around the tooth extraction site remained in ZOL-injected mice. The maxillary bone of FAM-ZOL-injected mice showed the strong fluorescence signal, while the remaining molars (M1, M2, M3) were absent of fluorescence. The visibly reduced fluorescence was observed in the tooth extraction sockets (*) and the adjacent palatal bone area (dotted line and arrowheads) B. Hematoxylin and eosin (H & E) stained histological sections of ZOL-injected mice showed significant inflammatory cell infiltration (Inflam) in the palatal/gingival soft tissue one week after tooth extraction. There were signs of abnormal epithelial hyperplasia (Epi.Hyp.: arrows). The extraction socket (*) exhibited new bone formation. TRAP staining revealed a number of osteoclasts (vertical arrows) on the surface of palatal bone (Palat.Bone) as well as in the extraction socket (*). TRAP+ multinuclear cells were found within the inflammation area (arrowheads). In non-decalcified cross-section of FAM-ZOL-injected mice, FAM-positive large cells (arrows) were found on the palatal bone as well as away from the bone surface (arrowheads). Taken together, these cells in the inflammatory area were thought to be ZOL-affected osteoclasts C. Immunohistology for cytokeratin 14 (CK14) was performed as described in the Materials and Method section, and confirmed oral epithelial hyperplasia in the palatal/gingival tissue of ZOL-injected mice (Epi.Hyp.: arrows). Epithelial hyperplasia was primarily observed within the strong inflammatory area D. Female B6 mice received IV injection of 500 μg/Kg ZOL or 0.9% NACL vehicle solution followed by maxillary first molar extraction after 6 days. Two weeks after tooth extraction the surface expression of CD45FITC+CD3PE or CD45FITC+DX5PE on cells obtained from gingival tissues. E. and bone marrow F. were assessed with flow cytometric analysis after staining with the respective PE- and FITC-conjugated antibodies. Isotype control antibodies were used as control. Right upper quadrant represent the percentage of CD3 and DX5 positive cells within the CD45 population. B6 mice underwent experimental treatment as described in Fig. 7D. Gingival tissues obtained from ZOL and NACL injected animals were digested as described in Materials and Method. Dissociated cells obtained from gingival tissues were cultured in the presence of IL-2 (10,000 units/ml). Supernatants were harvested 5 days after IL-2 treatment. The mean for (n = 4) is shown for each set in the figure. The difference between ZOL and NACL injected gingivae is significant at a P < 0.05 G. After euthanasia, femurs from ZOL and NACL injected animals were harvested and bone marrow cells were extracted and cultured (1 × 106 /ml) in the presence of IL-2 (10, 000 units/ml). Supernatants were harvested 5 days after IL-2 treatment. The mean for (n = 4) is shown for each set in the figure. The difference between ZOL and NACL injected bone marrow cells is significant at a P < 0.05 H. Gingival tissue, pancreas and adipose (1 × 106 /ml) from ZOL and NACL injected animals were cultured in the absence or presence of IL-2 (10, 000 units/ml) for 5 days, after which the supernatants were harvested and the levels of IFN-γ were measured with specific ELISA. *The difference between untreated or IL-2 stimulated cells from ZOL injected mice compared to untreated or IL-2 stimulated cells from NACL injected mice is significant at P < 0.05 I. Female WT B6 mice received IV injection of ZOL or NACL as described in Fig. 7A. After euthanasia, gingival tissues obtained from ZOL and NACL injected mice were digested as described in Materials and Methods section and cultured (1 × 106 /ml) in the presence of IL-2 (10, 000 units/ml). After five days of culture, supernatants from the cultures of gingival tissue were harvested and the levels of IL-6 secretion were measured by specific ELISA. J. Immunohistology for IL-6 was performed as described in the Materials and Methods section, and showed the IL-6 immuno-localization pattern was distinctly was different in osteoclasts in the jawbone of 0.9% NACL solution-injected control mice and ZOL-injected mice K. Female B6 mice received IV injection of ZOL or NACL as described in Fig. 7A. After euthanasia, femurs from ZOL and NACL injected mice were harvested and bone marrow cells were extracted and cultured (1 × 106 /ml) in the presence and absence of IL-2 (10,000 units/ml). After five days of culture, the supernatants were removed and the levels of IL-6 secretion were measured by specific ELISA. L. The pancreas was obtained from ZOL and NACL injected mice and digested as described in Materials and Methods section. Dissociated pancreatic cells were cultured in the presence and absence of IL-2 (10, 000 units/ml). After five days of culture, the supernatants were removed and the levels of IL-6 secretion were measured by specific ELISA M. Dissociated gingival, pancreas and adipose cells (1 × 106 /ml) from ZOL and NACL injected animals were cultured in the absence or presence of IL-2 (10, 000 units/ml) for 5 days and used as effectors against 51Cr labeled ST63 cells at various effector to target ratios in a standard 4 hour 51Cr release assay. The lytic units 30/106 cells were determined using inverse number of NK cells required to lyse 30% of the ST63 cells X100 N. Total cells from the bone marrow were prepared as described in Fig. 7K O. were prepared as described in Fig. 7K. NK cells were purified from bone marrow cells and cultured in the presence or absence of IL-2 (10, 000 units/ml) for 5 days P. Afterwards, the cells were used as effectors against 51Cr labeled ST63 cells at various effector to target ratios in a standard 4 hour 51Cr release assay. The lytic units 30/106 cells were determined using inverse number of NK cells required to lyse 30% of ST63 cells X100. * The difference between IL-2 treated BM (N) or BM-NK (O) cells obtained from mice injected with ZOL compared to IL-2 treated BM or BM-NK cells from mice injected with NACL is significant at P < 0.05. NK cells purified from bone marrow were cultured in the presence and absence of IL-2 (10, 000 units/ml) after which supernatants were harvested at day 5 and the levels of IFN-γ Q. and IL-6 R. were determined by specific ELISAs. One week after tooth extraction, total RNA samples were isolated from palatal/gingival tissue of control and ZOL-injected mice. PCR microarray analysis revealed the suppressed expression of pro-inflammatory cytokines such as IFN-γ (ifng), IL-6, -12b, -15, -2 and TNF-α (Tnf) in the ZOL-injected mice. Those unaffected cytokines included anti-inflammatory cytokines such as IL-10 and -4 (R) (A complete list of PCR microarray data can be found in Fig. S3).
Figure 7
Figure 7. In vivo injection of ZOL triggered significant IFN-γ and IL-6 secretion by bone marrow-derived cells but inhibited secretion by gingival cells
A. Female Balb/c mice received IV injection of 500 μg/Kg ZOL or 0.9% NACL vehicle solution followed by maxillary first molar extraction after 6 days. Two weeks after tooth extraction, the maxillary palatal mucosa of 0.9% NACL solution-injected control mice showed complete wound healing, whereas tissue swelling (dotted line) around the tooth extraction site remained in ZOL-injected mice. The maxillary bone of FAM-ZOL-injected mice showed the strong fluorescence signal, while the remaining molars (M1, M2, M3) were absent of fluorescence. The visibly reduced fluorescence was observed in the tooth extraction sockets (*) and the adjacent palatal bone area (dotted line and arrowheads) B. Hematoxylin and eosin (H & E) stained histological sections of ZOL-injected mice showed significant inflammatory cell infiltration (Inflam) in the palatal/gingival soft tissue one week after tooth extraction. There were signs of abnormal epithelial hyperplasia (Epi.Hyp.: arrows). The extraction socket (*) exhibited new bone formation. TRAP staining revealed a number of osteoclasts (vertical arrows) on the surface of palatal bone (Palat.Bone) as well as in the extraction socket (*). TRAP+ multinuclear cells were found within the inflammation area (arrowheads). In non-decalcified cross-section of FAM-ZOL-injected mice, FAM-positive large cells (arrows) were found on the palatal bone as well as away from the bone surface (arrowheads). Taken together, these cells in the inflammatory area were thought to be ZOL-affected osteoclasts C. Immunohistology for cytokeratin 14 (CK14) was performed as described in the Materials and Method section, and confirmed oral epithelial hyperplasia in the palatal/gingival tissue of ZOL-injected mice (Epi.Hyp.: arrows). Epithelial hyperplasia was primarily observed within the strong inflammatory area D. Female B6 mice received IV injection of 500 μg/Kg ZOL or 0.9% NACL vehicle solution followed by maxillary first molar extraction after 6 days. Two weeks after tooth extraction the surface expression of CD45FITC+CD3PE or CD45FITC+DX5PE on cells obtained from gingival tissues. E. and bone marrow F. were assessed with flow cytometric analysis after staining with the respective PE- and FITC-conjugated antibodies. Isotype control antibodies were used as control. Right upper quadrant represent the percentage of CD3 and DX5 positive cells within the CD45 population. B6 mice underwent experimental treatment as described in Fig. 7D. Gingival tissues obtained from ZOL and NACL injected animals were digested as described in Materials and Method. Dissociated cells obtained from gingival tissues were cultured in the presence of IL-2 (10,000 units/ml). Supernatants were harvested 5 days after IL-2 treatment. The mean for (n = 4) is shown for each set in the figure. The difference between ZOL and NACL injected gingivae is significant at a P < 0.05 G. After euthanasia, femurs from ZOL and NACL injected animals were harvested and bone marrow cells were extracted and cultured (1 × 106 /ml) in the presence of IL-2 (10, 000 units/ml). Supernatants were harvested 5 days after IL-2 treatment. The mean for (n = 4) is shown for each set in the figure. The difference between ZOL and NACL injected bone marrow cells is significant at a P < 0.05 H. Gingival tissue, pancreas and adipose (1 × 106 /ml) from ZOL and NACL injected animals were cultured in the absence or presence of IL-2 (10, 000 units/ml) for 5 days, after which the supernatants were harvested and the levels of IFN-γ were measured with specific ELISA. *The difference between untreated or IL-2 stimulated cells from ZOL injected mice compared to untreated or IL-2 stimulated cells from NACL injected mice is significant at P < 0.05 I. Female WT B6 mice received IV injection of ZOL or NACL as described in Fig. 7A. After euthanasia, gingival tissues obtained from ZOL and NACL injected mice were digested as described in Materials and Methods section and cultured (1 × 106 /ml) in the presence of IL-2 (10, 000 units/ml). After five days of culture, supernatants from the cultures of gingival tissue were harvested and the levels of IL-6 secretion were measured by specific ELISA. J. Immunohistology for IL-6 was performed as described in the Materials and Methods section, and showed the IL-6 immuno-localization pattern was distinctly was different in osteoclasts in the jawbone of 0.9% NACL solution-injected control mice and ZOL-injected mice K. Female B6 mice received IV injection of ZOL or NACL as described in Fig. 7A. After euthanasia, femurs from ZOL and NACL injected mice were harvested and bone marrow cells were extracted and cultured (1 × 106 /ml) in the presence and absence of IL-2 (10,000 units/ml). After five days of culture, the supernatants were removed and the levels of IL-6 secretion were measured by specific ELISA. L. The pancreas was obtained from ZOL and NACL injected mice and digested as described in Materials and Methods section. Dissociated pancreatic cells were cultured in the presence and absence of IL-2 (10, 000 units/ml). After five days of culture, the supernatants were removed and the levels of IL-6 secretion were measured by specific ELISA M. Dissociated gingival, pancreas and adipose cells (1 × 106 /ml) from ZOL and NACL injected animals were cultured in the absence or presence of IL-2 (10, 000 units/ml) for 5 days and used as effectors against 51Cr labeled ST63 cells at various effector to target ratios in a standard 4 hour 51Cr release assay. The lytic units 30/106 cells were determined using inverse number of NK cells required to lyse 30% of the ST63 cells X100 N. Total cells from the bone marrow were prepared as described in Fig. 7K O. were prepared as described in Fig. 7K. NK cells were purified from bone marrow cells and cultured in the presence or absence of IL-2 (10, 000 units/ml) for 5 days P. Afterwards, the cells were used as effectors against 51Cr labeled ST63 cells at various effector to target ratios in a standard 4 hour 51Cr release assay. The lytic units 30/106 cells were determined using inverse number of NK cells required to lyse 30% of ST63 cells X100. * The difference between IL-2 treated BM (N) or BM-NK (O) cells obtained from mice injected with ZOL compared to IL-2 treated BM or BM-NK cells from mice injected with NACL is significant at P < 0.05. NK cells purified from bone marrow were cultured in the presence and absence of IL-2 (10, 000 units/ml) after which supernatants were harvested at day 5 and the levels of IFN-γ Q. and IL-6 R. were determined by specific ELISAs. One week after tooth extraction, total RNA samples were isolated from palatal/gingival tissue of control and ZOL-injected mice. PCR microarray analysis revealed the suppressed expression of pro-inflammatory cytokines such as IFN-γ (ifng), IL-6, -12b, -15, -2 and TNF-α (Tnf) in the ZOL-injected mice. Those unaffected cytokines included anti-inflammatory cytokines such as IL-10 and -4 (R) (A complete list of PCR microarray data can be found in Fig. S3).

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