Tissue-specific cues are critical for homeostasis at mucosal barriers. Here, we report that the clotting factor fibrin is a critical regulator of neutrophil function at the oral mucosal barrier. We demonstrate that commensal microbiota trigger extravascular fibrin deposition in the oral mucosa. Fibrin engages neutrophils through the α
Chronic granulomatous disease (CGD) is caused by defects in nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) complex subunits (gp91 phox (a.k.a. Nox2), p47 phox , p67 phox , p22 phox , p40 phox ) leading to reduced phagocyte-derived reactive oxygen species production. Almost half of patients with CGD develop inflammatory bowel disease, and the involvement of the intestinal microbiome in relation to this predisposing immunodeficiency has not been explored. Although CGD mice do not spontaneously develop colitis, we demonstrate that p47 phox−/− mice have increased susceptibility to dextran sodium sulfate colitis in association with a distinct colonic transcript and microbiome signature. Neither restoring NOX2 reactive oxygen species production nor normalizing the microbiome using cohoused adult p47 phox−/− with B6Tac (wild type) mice reversed this phenotype. However, breeding p47 phox+/− mice and standardizing the microflora between littermate p47 phox−/− and B6Tac mice from birth significantly reduced dextran sodium sulfate colitis susceptibility in p47 phox−/− mice. We found similarly decreased colitis susceptibility in littermate p47 phox−/− and B6Tac mice treated with Citrobacter rodentium. Our findings suggest that the microbiome signature established at birth may play a bigger role than phagocyte-derived reactive oxygen species in mediating colitis susceptibility in CGD mice. These data further support bacteria-related disease in CGD colitis.
Aggregatibacter actinomycetemcomitans generates citrullinated autoantigens involved in rheumatoid arthritis through its pore-forming toxin leukotoxin A.
Abstract N eutrophil E xtracellular T rap (NET) formation (NETosis) is a unique process that occurs in response to numerous stimuli. To investigate NETosis, we created a method that can be used easily without the need for complex programming abilities and commercial software packages. This article describes a fully automated assay to quantify NETosis using fluorescence live imaging on an automated widefield inverted microscope. Herein, we describe (1) sample preparation, (2) required equipment for automated acquisition, and finally (3) analysis of NETosis using the readily available image analysis software Fiji (ImageJ2). This protocol can be adapted to evaluate NETosis after different stimuli, and can be easily modified to allow high‐throughput acquisition and analysis using a multi‐well plate format. Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : Neutrophil isolation and plate setup Basic Protocol 2 : Microscope and acquisition setup for automated high throughput imaging Basic Protocol 3 : Analysis of NETosis and apoptosis data
Immunodeficiency, the consequence of HIV-1 infection, predisposes the host to opportunistic infections. In turn, opportunistic pathogens influence target cell susceptibility to HIV-1 infection and replication. Although the advent of highly active antiretroviral therapy (HAART) has altered these sequelae, co-infections may prevail in some parts of the world and in failed HAART regimens. Moreover, immune activation as occurs in tonsil and non-infectious mucosal inflammatory lesions may also be associated with proximal sites of viral replication. These connections between enhancement of HIV-1 infection and activation/inflammation warrant further elucidation of the factors promoting permissiveness to HIV-1 infection. Using the opportunistic pathogen Mycobacterium avium as an in vitro model, we demonstrated that co-infection facilitated HIV-1 infection of monocyte-macrophages by multiple pathways. M. avium activated NF-kappaB, the downstream consequences of which included augmented expression of tumor necrosis factor alpha and CCR5 receptors, both permissive for sustaining HIV-1 infection. Pronounced viral replication in lymph nodes co-infected with M. avium and HIV-1 paralleled these in vitro findings. Furthermore, reduction in viral burden is associated with treatment of infected or inflamed tissues, underscoring the link between immune activation and viral replication.
We present the first, to our knowledge, described case of carcinosarcoma of the maxilla in a squirrel monkey. Carcinosarcomas are rare tumors of the upper aerodigestive tract, and consist of carcinomatous and sarcomatous tissue. Histologic analysis revealed a neoplasm composed of an adenocarcinomatous component (epithelial element) and a mesenchymal component (sarcomatous element). Metastatic growth was documented in the lung tissue and the submandibular lymph node. The histolopathologic findings, the pattern of metastasis, and the clinical progression closely resembled those of carcinosarcoma involving salivary glands in humans.
Differential expression of secretory leukocyte protease inhibitor (SLPI) impacts on tumor progression. SLPI directly inhibits elastase and other serine proteases, and regulates matrix metalloproteinases, plasminogen activation, and plasmin downstream targets to influence invasion. We examined tissues from human oral squamous cell carcinoma (OSCC) for SLPI expression in parallel with proteases associated with tumor progression and evaluated their relationships using tumor cell lines. Significantly decreased SLPI was detected in OSCC compared to normal oral epithelium. Furthermore, an inverse correlation between SLPI and histological parameters associated with tumor progression, including stage of invasion, pattern of invasion, invasive cell grade, and composite histological tumor score was evident. Conversely, elevated plasmin and elastase were positively correlated with histological parameters of tumor invasion. In addition to its known inhibition of elastase, we identify SLPI as a novel inhibitor of plasminogen activation through its interaction with annexin A2 with concomitant reduced plasmin generation by macrophages and OSCC cell lines. In an in vitro assay measuring invasive activity, SLPI blocked protease-dependent tumor cell migration. Our data suggest that SLPI may possess antitumorigenic activity by virtue of its ability to interfere with multiple requisite proteolytic steps underlying tumor cell invasion and may provide insight into potential stratification of oral cancer according to risk of occult metastasis, guiding treatment strategies. Differential expression of secretory leukocyte protease inhibitor (SLPI) impacts on tumor progression. SLPI directly inhibits elastase and other serine proteases, and regulates matrix metalloproteinases, plasminogen activation, and plasmin downstream targets to influence invasion. We examined tissues from human oral squamous cell carcinoma (OSCC) for SLPI expression in parallel with proteases associated with tumor progression and evaluated their relationships using tumor cell lines. Significantly decreased SLPI was detected in OSCC compared to normal oral epithelium. Furthermore, an inverse correlation between SLPI and histological parameters associated with tumor progression, including stage of invasion, pattern of invasion, invasive cell grade, and composite histological tumor score was evident. Conversely, elevated plasmin and elastase were positively correlated with histological parameters of tumor invasion. In addition to its known inhibition of elastase, we identify SLPI as a novel inhibitor of plasminogen activation through its interaction with annexin A2 with concomitant reduced plasmin generation by macrophages and OSCC cell lines. In an in vitro assay measuring invasive activity, SLPI blocked protease-dependent tumor cell migration. Our data suggest that SLPI may possess antitumorigenic activity by virtue of its ability to interfere with multiple requisite proteolytic steps underlying tumor cell invasion and may provide insight into potential stratification of oral cancer according to risk of occult metastasis, guiding treatment strategies. Head and neck squamous cell carcinoma is among the top 10 most frequent cancers in the US.1Jemal A. Siegel R. Ward E. Hao Y. Xu J. Murray T. Thun M.J. Cancer statistics, 2008.CA Cancer J Clin. 2008; 58: 71-96Crossref PubMed Scopus (10141) Google Scholar Unfavorable prognosis is mainly due to local invasion and frequent spread to lymph nodes (LN).2Fukano H. Matsuura H. Hasegawa Y. Nakamura S. Depth of invasion as a predictive factor for cervical lymph node metastasis in tongue carcinoma.Head Neck. 1997; 19: 205-210Crossref PubMed Google Scholar Generally, the invasive property of tumor cells is dependent on activation of proteases, such as plasmin at the cell surface.3Curino A. Patel V. Nielsen B.S. Iskander A.J. Ensley J.F. Yoo G.H. Holsinger F.C. Myers J.N. El-Nagaar A. Kellman R.M. Shillitoe E.J. Molinolo A.A. Gutkind J.S. Bugge T.H. Detection of plasminogen activators in oral cancer by laser capture microdissection combined with zymography.Oral Oncol. 2004; 40: 1026-1032Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 4Ranson M. Andronicos N.M. Plasminogen binding and cancer: promises and pitfalls.Front Biosci. 2003; 8: S294-S304Crossref PubMed Scopus (54) Google Scholar, 5Tang C.H. Wei Y. The urokinase receptor and integrins in cancer progression.Cell Mol Life Sci. 2008; 65: 1916-1932Crossref PubMed Scopus (53) Google Scholar Inactive plasminogen is converted to the active, broad-spectrum serine proteinase, plasmin, by plasminogen activators (PA), including urokinase-type PA (uPA) and tissue-type PA (tPA), and their overexpression is considered central in malignant tumor progression.6Duffy M.J. The urokinase plasminogen activator system: role in malignancy.Curr Pharm Des. 2004; 10: 39-49Crossref PubMed Scopus (339) Google Scholar, 7Sharma M.C. Sharma M. The role of annexin II in angiogenesis and tumor progression: a potential therapeutic target.Curr Pharm Des. 2007; 13: 3568-3575Crossref PubMed Scopus (119) Google Scholar Efficient plasmin generation involves plasminogen binding, together with PA, on cell surfaces by annexin A2, urokinase-type PA receptor, and other docking sites that colocalize enzyme and substrate.8Kwon M. MacLeod T.J. Zhang Y. Waisman D.M. S100A10, annexin A2, and annexin a2 heterotetramer as candidate plasminogen receptors.Front Biosci. 2005; 10: 300-325Crossref PubMed Scopus (149) Google Scholar, 9Brownstein C. Falcone D.J. Jacovina A. Hajjar K.A. A mediator of cell surface-specific plasmin generation.Ann N Y Acad Sci. 2001; 947 (discussion 155–146): 143-155Crossref PubMed Scopus (31) Google Scholar, 10Shi Z. Stack M.S. Urinary-type plasminogen activator (uPA) and its receptor (uPAR) in squamous cell carcinoma of the oral cavity.Biochem J. 2007; 407: 153-159Crossref PubMed Scopus (46) Google Scholar Loss of annexin A2 from surfaces of cancer cells results in significantly reduced plasmin.11Diaz V.M. Hurtado M. Thomson T.M. Reventos J. Paciucci R. Specific interaction of tissue-type plasminogen activator (t-PA) with annexin II on the membrane of pancreatic cancer cells activates plasminogen and promotes invasion in vitro.Gut. 2004; 53: 993-1000Crossref PubMed Scopus (105) Google Scholar Plasmin, in turn, activates matrix-degrading metalloproteinases (MMPs) produced by tumor cells as well as by stromal cells.12Pepper M.S. Role of the matrix metalloproteinase and plasminogen activator-plasmin systems in angiogenesis.Arterioscler Thromb Vasc Biol. 2001; 21: 1104-1117Crossref PubMed Scopus (672) Google Scholar Invasive properties of tumor cells are aided and abetted by infiltrating inflammatory cells, particularly tumor-associated macrophages, responsible for release of growth factors and matrix destructive enzymes.13Mantovani A. Sica A. Allavena P. Garlanda C. Locati M. Tumor-associated macrophages and the related myeloid-derived suppressor cells as a paradigm of the diversity of macrophage activation.Hum Immunol. 2009; 70: 325-330Crossref PubMed Scopus (265) Google Scholar Dysregulation of the protease–antiprotease balance underlies tumor cell invasion, warranting examination of potential deficiencies in antiproteases as contributors to metastatic events. Recently, the antiprotease secretory leukocyte protease inhibitor (SLPI), a member of the whey acidic protein family, has been implicated in tumorigenesis.14Devoogdt N. Revets H. Ghassabeh G.H. De Baetselier P. Secretory leukocyte protease inhibitor in cancer development.Ann N Y Acad Sci. 2004; 1028: 380-389Crossref PubMed Scopus (45) Google Scholar, 15Bouchard D. Morisset D. Bourbonnais Y. Tremblay G.M. Proteins with whey-acidic-protein motifs and cancer.Lancet Oncol. 2006; 7: 167-174Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar SLPI is a 11.7-kDa nonglycosylated epithelial-derived protein that neutralizes elastase, tryptase, and cathepsin G. Elastase, capable of splitting a variety of structural and functional proteins, also activates MMP, leading to amplified extracellular matrix cleavage.16Song X. Zeng L. Jin W. Thompson J. Mizel D.E. Lei K. Billinghurst R.C. Poole A.R. Wahl S.M. Secretory leukocyte protease inhibitor suppresses the inflammation and joint damage of bacterial cell wall-induced arthritis.J Exp Med. 1999; 190: 535-542Crossref PubMed Scopus (127) Google Scholar, 17Kelly E. Greene C.M. McElvaney N.G. Targeting neutrophil elastase in cystic fibrosis.Expert Opin Ther Targets. 2008; 12: 145-157Crossref PubMed Scopus (65) Google Scholar Moreover, SLPI directly inhibits macrophage MMP, possibly through inhibition of NFκB,18Zhang Y. DeWitt D.L. McNeely T.B. Wahl S.M. Wahl L.M. Secretory leukocyte protease inhibitor suppresses the production of monocyte prostaglandin H synthase-2, prostaglandin E2, and matrix metalloproteinases.J Clin Invest. 1997; 99: 894-900Crossref PubMed Scopus (160) Google Scholar, 19Taggart C.C. Cryan S.A. Weldon S. Gibbons A. Greene C.M. Kelly E. Low T.B. O'Neill S.J. McElvaney N.G. Secretory leucoprotease inhibitor binds to NF-kappaB binding sites in monocytes and inhibits p65 binding.J Exp Med. 2005; 202: 1659-1668Crossref PubMed Scopus (166) Google Scholar and absence of SLPI is associated with enhanced elastase activity, delayed matrix accumulation, and aberrant healing.16Song X. Zeng L. Jin W. Thompson J. Mizel D.E. Lei K. Billinghurst R.C. Poole A.R. Wahl S.M. Secretory leukocyte protease inhibitor suppresses the inflammation and joint damage of bacterial cell wall-induced arthritis.J Exp Med. 1999; 190: 535-542Crossref PubMed Scopus (127) Google Scholar, 20Ashcroft G.S. Lei K. Jin W. Longenecker G. Kulkarni A.B. Greenwell-Wild T. Hale-Donze H. McGrady G. Song X.Y. Wahl S.M. Secretory leukocyte protease inhibitor mediates non-redundant functions necessary for normal wound healing.Nat Med. 2000; 6: 1147-1153Crossref PubMed Scopus (340) Google Scholar, 21Zhu J. Nathan C. Jin W. Sim D. Ashcroft G.S. Wahl S.M. Lacomis L. Erdjument-Bromage H. Tempst P. Wright C.D. Ding A. Conversion of proepithelin to epithelins: roles of SLPI and elastase in host defense and wound repair.Cell. 2002; 111: 867-878Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar, 22Angelov N. Moutsopoulos N. Jeong M.J. Nares S. Ashcroft G. Wahl S.M. Aberrant mucosal wound repair in the absence of secretory leukocyte protease inhibitor.Thromb Haemost. 2004; 92: 288-297PubMed Google Scholar Due to its generation by epithelial cells and its unique repertoire of antiproteolytic, antimicrobial, and anti-inflammatory properties,20Ashcroft G.S. Lei K. Jin W. Longenecker G. Kulkarni A.B. Greenwell-Wild T. Hale-Donze H. McGrady G. Song X.Y. Wahl S.M. Secretory leukocyte protease inhibitor mediates non-redundant functions necessary for normal wound healing.Nat Med. 2000; 6: 1147-1153Crossref PubMed Scopus (340) Google Scholar, 21Zhu J. Nathan C. Jin W. Sim D. Ashcroft G.S. Wahl S.M. Lacomis L. Erdjument-Bromage H. Tempst P. Wright C.D. Ding A. Conversion of proepithelin to epithelins: roles of SLPI and elastase in host defense and wound repair.Cell. 2002; 111: 867-878Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar, 22Angelov N. Moutsopoulos N. Jeong M.J. Nares S. Ashcroft G. Wahl S.M. Aberrant mucosal wound repair in the absence of secretory leukocyte protease inhibitor.Thromb Haemost. 2004; 92: 288-297PubMed Google Scholar, 23Ma G. Greenwell-Wild T. Lei K. Jin W. Swisher J. Hardegen N. Wild C.T. Wahl S.M. Secretory leukocyte protease inhibitor binds to annexin II, a cofactor for macrophage HIV-1 infection.J Exp Med. 2004; 200: 1337-1346Crossref PubMed Scopus (172) Google Scholar, 24McNeely T.B. Dealy M. Dripps D.J. Orenstein J.M. Eisenberg S.P. Wahl S.M. Secretory leukocyte protease inhibitor: a human saliva protein exhibiting anti-human immunodeficiency virus 1 activity in vitro.J Clin Invest. 1995; 96: 456-464Crossref PubMed Scopus (369) Google Scholar, 25Malamud D. Wahl S.M. The mouth: a gateway or a trap for HIV?.AIDS. 2010; 24: 5-16Crossref PubMed Scopus (27) Google Scholar, 26Sallenave J.M. Secretory leukocyte protease inhibitor and elafin/trappin-2: versatile mucosal antimicrobials and regulators of immunity.Am J Respir Cell Mol Biol. 2010; 42: 635-643Crossref PubMed Scopus (98) Google Scholar SLPI expression has been examined in multiple tumor types. Nonetheless, its role in tumorigenesis remains controversial, since depending on cancer type, either up-regulation or down-regulation has been observed.14Devoogdt N. Revets H. Ghassabeh G.H. De Baetselier P. Secretory leukocyte protease inhibitor in cancer development.Ann N Y Acad Sci. 2004; 1028: 380-389Crossref PubMed Scopus (45) Google Scholar, 15Bouchard D. Morisset D. Bourbonnais Y. Tremblay G.M. Proteins with whey-acidic-protein motifs and cancer.Lancet Oncol. 2006; 7: 167-174Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar Correlations between reduced SLPI and tumor spread have suggested antitumorigenic functions,27Tian X. Shigemasa K. Hirata E. Gu L. Uebaba Y. Nagai N. O'Brien T.J. Ohama K. Expression of human kallikrein 7 (hK7/SCCE) and its inhibitor antileukoprotease (ALP/SLPI) in uterine endocervical glands and in cervical adenocarcinomas.Oncol Rep. 2004; 12: 1001-1006PubMed Google Scholar and as a potentially protective molecule against liver metastasis28Wang N. Thuraisingam T. Fallavollita L. Ding A. Radzioch D. Brodt P. The secretory leukocyte protease inhibitor is a type 1 insulin-like growth factor receptor-regulated protein that protects against liver metastasis by attenuating the host proinflammatory response.Cancer Res. 2006; 66: 3062-3070Crossref PubMed Scopus (49) Google Scholar and mammary tumor invasion. However, SLPI reportedly promotes invasion-independent metastasis,29Sugino T. Yamaguchi T. Ogura G. Kusakabe T. Goodison S. Homma Y. Suzuki T. The secretory leukocyte protease inhibitor (SLPI) suppresses cancer cell invasion but promotes blood-borne metastasis via an invasion-independent pathway.J Pathol. 2007; 212: 152-160Crossref PubMed Scopus (32) Google Scholar proliferation, and survival in ovarian cancer,30Simpkins F.A. Devoogdt N.M. Rasool N. Tchabo N.E. Alejandro E.U. Kamrava M.M. Kohn E.C. The alarm anti-protease, secretory leukocyte protease inhibitor, is a proliferation and survival factor for ovarian cancer cells.Carcinogenesis. 2008; 29: 466-472Crossref PubMed Scopus (32) Google Scholar providing a far from clear view of its role in tumor development and progression. To define potential contributions of SLPI to head and neck squamous cell carcinomas (HNSCC), we examined tumors for expression of SLPI as well as downstream proteases in tumors that did or did not progress to regional lymph node metastasis. SLPI expression in oral squamous cell carcinoma (OSCC) was significantly lower than in normal oral epithelium and exhibited significant inverse correlations with invasion parameters. Further characterization of its potential mechanism of action revealed a novel inhibitory role for SLPI in plasminogen activation on the cell surface, where it binds to annexin A2 to intercede in tPA activation of plasminogen, and in an in vitro tumor model, SLPI inhibits invasive activity. Our results provide evidence for a potential protective role of SLPI in oral cancer, and as a tool for possible stratification of oral cancer according to risk of occult metastasis, possibly guiding treatment decisions and the need for elective neck dissection in a clinically negative neck. Patients diagnosed with early stage, clinically negative (N0) neck (stage I: T1N0M0 or stage II: T2N0M0) OSCC of the tongue or floor of the mouth and treated at the University of Maryland between 1994 and 2000 were identified (Institutional Review Board, University of Maryland; exemption #NN-040301, #H27950).31Warburton G. Nikitakis N.G. Roberson P. Marinos N.J. Wu T. Sauk Jr., J.J. Ord R.A. Wahl S.M. Histopathological and lymphangiogenic parameters in relation to lymph node metastasis in early stage oral squamous cell carcinoma.J Oral Maxillofac Surg. 2007; 65: 475-484Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar Only patients treated by primary tumor resection without neck dissection or irradiation were included. The occurrence of delayed neck metastasis (DNM) to regional LN was recorded, with DNM+ and DNM− signifying presence or absence of LN metastasis, respectively, within the 5-year assessment. DNM represents occult LN metastatic disease that is clinically and radiographically undetectable at the time of diagnosis and treatment, but later becomes apparent.31Warburton G. Nikitakis N.G. Roberson P. Marinos N.J. Wu T. Sauk Jr., J.J. Ord R.A. Wahl S.M. Histopathological and lymphangiogenic parameters in relation to lymph node metastasis in early stage oral squamous cell carcinoma.J Oral Maxillofac Surg. 2007; 65: 475-484Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar Formalin-fixed, paraffin-embedded tissue samples from 24 patients were obtained from the pathology archive, and oral mucosal tissues from healthy subjects (n = 5) were evaluated as controls. Representative surgical tumor sections were reviewed by two oral and maxillofacial pathologists (N.G.N., R.C.) blinded to LN status. Tumors were classified based on: i) degree of keratinization; ii) nuclear polymorphism; iii) number of mitoses; iv) invasive pattern; v) stage of invasion; and vi) lymphoplasmacytic infiltration, paying particular attention to the most dysplastic area of the invasive front.31Warburton G. Nikitakis N.G. Roberson P. Marinos N.J. Wu T. Sauk Jr., J.J. Ord R.A. Wahl S.M. Histopathological and lymphangiogenic parameters in relation to lymph node metastasis in early stage oral squamous cell carcinoma.J Oral Maxillofac Surg. 2007; 65: 475-484Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 32Anneroth G. Batsakis J. Luna M. Review of the literature and a recommended system of malignancy grading in oral squamous cell carcinomas.Scand J Dent Res. 1987; 95: 229-249PubMed Google Scholar Each parameter was graded for individual tumor sections using a four-point rating scale, with grade 1 corresponding to a more differentiated presentation (eg, abundant keratinization), and grade 4 to the least differentiated presentation (eg, absence of keratinization). A composite grade (CG) was calculated as the sum of six individual grades. In addition, the invasive cell grade (ICG), another widely used multifactorial and semiquantitative grading system linked to occult metastasis,33Russolo M. Giacomarra V. Papanikolla L. Tirelli G. Prognostic indicators of occult metastases in oral cancer.Laryngoscope. 2002; 112: 1320-1323Crossref PubMed Scopus (15) Google Scholar was calculated as the sum of five parameters, excluding stage of invasion. Tumor thickness/depth of invasion was measured microscopically in millimeters31Warburton G. Nikitakis N.G. Roberson P. Marinos N.J. Wu T. Sauk Jr., J.J. Ord R.A. Wahl S.M. Histopathological and lymphangiogenic parameters in relation to lymph node metastasis in early stage oral squamous cell carcinoma.J Oral Maxillofac Surg. 2007; 65: 475-484Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar from the surface of the epithelium to deepest invading tumor island or cell, using a reconstructed line excluding exophytic tumor components and including thickness of epithelium lost due to ulceration. Tissue sections, treated for epitope retrieval and to block endogenous peroxidase, were blocked with corresponding pre-immune serum or FBS-Tween 20 and incubated overnight at 4°C with primary antibodies: anti-SLPI (R&D Systems, Minneapolis, MN) 1:100; anti-plasmin (Genway Biotech, San Diego, CA) 1:50; anti-elastase (DakoCytomation, Glostrup, Denmark) 1:50; anti-MMP9 (Chemicon, Temecula, CA); or isotype control. After PBS washes, immunolabeling was detected using a biotinylated secondary antibody followed by avidin-biotin horseradish peroxidase (Zymed, Grand Island, NY) and diaminobenzidine, and counterstain with hematoxylin. Immunostaining was assessed (N.G.N., R.C.) and classified according to percentage stained cells and intensity of stain. Immunohistochemical (IHC) staining was evaluated for tumor cells and/or for inflammatory infiltrates using a four-point score: a score of 0 = <1% positive cells, 1 = 1% to 25%, 2 = 26% to 50%, 3 = 51% to 75%, and 4 = 76% to 100%. In some cases, a six-point scale further refined the level of expression: 0 = <1% positive, with 1 (1% to 10%), 2 (11% to 30%), 3 (31% to 50%), 4 (51% to 70%), 5 (71% to 90%), and 6 (≥91%) representing increasing positivity. Intensity of staining was classified as weak (W), moderate (M), or strong (S). The HNSCC cell line Tu1386 (kind gift from Dr. P. Sacks, New York University)34Sacks P.G. Cell, tissue and organ culture as in vitro models to study the biology of squamous cell carcinomas of the head and neck.Cancer Metastasis Rev. 1996; 15: 27-51Crossref PubMed Scopus (102) Google Scholar and HN12 cells, derived from metastatic SCC35Yeudall W.A. Miyazaki H. Ensley J.F. Cardinali M. Gutkind J.S. Patel V. Uncoupling of epidermal growth factor-dependent proliferation and invasion in a model of squamous carcinoma progression.Oral Oncol. 2005; 41: 698-708Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar (a kind gift from Dr. J. Silvio Gutkind, National Institute of Dental and Craniofacial Research), were maintained in Dulbecco's Modified Eagle's Medium (DMEM) (Mediatech, Inc, Herndon, VA) containing 10% FBS in a humidified (37°C, 5% CO2) incubator. Normal human keratinocyte cell line (NHEK-Ad) (Clonetics, San Diego, CA) was cultured in keratinocyte basal medium-2 (Clonetics). For experimental assays, cells were trypsinized (Accutase; Innovative Cell Technologies, San Diego, CA) and harvested on reaching 80% to 90% confluence. MMP2 activity was measured using Calbiochem InnoZyme Gelatinase Activity Assay Kit (Calbiochem/Merck, Darmstadt, Germany) containing a triple-helical, collagen-like, fluorogenic substrate. In brief, cell lines (2.0 × 105) grown to confluency in 12-well plates were cultured in serum-free medium overnight and SLPI (0 to 10 μg/mL) added before supernatants were collected (6 to 24 hours) and concentrated (Microcon; Millipore Corporation, Bedford, MA). After adding 90 μL of concentrated sample and 10 μL of substrate working solution to 96-well plates, the plates were incubated at 37°C for 2 to 6 hours and read with a fluorescence plate reader. Cleavage of substrate results in increased fluorescence at 320-nm excitation and emission wavelength of 405 nm. Human peripheral blood mononuclear cells were obtained by leukapheresis of healthy volunteers (Department of Transfusion Medicine, National Institutes of Health). Monocytes were purified by elutriation24McNeely T.B. Dealy M. Dripps D.J. Orenstein J.M. Eisenberg S.P. Wahl S.M. Secretory leukocyte protease inhibitor: a human saliva protein exhibiting anti-human immunodeficiency virus 1 activity in vitro.J Clin Invest. 1995; 96: 456-464Crossref PubMed Scopus (369) Google Scholar and cultured in DMEM supplemented with 2 mmol/L l-glutamine, 100 μl/mL penicillin, and 100 μg/mL streptomycin (Sigma, St. Louis, MO). For macrophages, monocytes were adhered and cultured in supplemented DMEM with 10% FBS for 7 to 10 days.24McNeely T.B. Dealy M. Dripps D.J. Orenstein J.M. Eisenberg S.P. Wahl S.M. Secretory leukocyte protease inhibitor: a human saliva protein exhibiting anti-human immunodeficiency virus 1 activity in vitro.J Clin Invest. 1995; 96: 456-464Crossref PubMed Scopus (369) Google Scholar Macrophages (1 × 106 cells/mL, 100 μL/tube) in buffer (HEPES-buffered saline containing 3 mmol/L CaCl2 and 1 mmol/L MgCl2; Mediatech, Inc.) were incubated with 100 nmol/L N-terminal glutamic acid plasminogen (glu-plasminogen) (American Diagnostica, Greenwich, CT) for 1 hour at 4°C. Then rabbit polyclonal anti-annexin II (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit IgG (60 μg/mL; Jackson ImmunoResearch Laboratories, West Grove, PA), or SLPI (1 to 25 μg/mL) was added for 45 minutes at 4°C. The cells were washed with buffer, and both tPA (12 nmol/L; Calbiochem) and fluorogenic plasmin substrate AFC-081(166 μmol/L, d-valine-leucine-lysine-7-amino-4-trifluoromethyl coumarin; Enzyme Systems Products, Aurora, OH) were added and substrate hydrolysis measured at 5-minute intervals (400-nm excitation and 505-nm emission).36Hajjar K.A. Jacovina A.T. Chacko J. An endothelial cell receptor for plasminogen/tissue plasminogen activator. I. Identity with annexin II.J Biol Chem. 1994; 269: 21191-21197Abstract Full Text PDF PubMed Google Scholar HN12, Tu1386, and NHEK cells in 12-well plates (2 × 105 cells/mL) were incubated with SLPI (5 μg/mL) or ε-amino-caproic acid (ACA; Calbiochem) for 1 hour in phenol red–free and serum-free DMEM and 0.5 μmol/L purified glu-plasminogen added for 2 to 4 hours. Kinetics of constitutive cell-mediated plasminogen activation was determined by measuring amidolytic activity of plasmin after the reaction was initiated by addition of substrate H-d-norleucyl-hexahydrotyrosyl-lysine-p-nitroanilide (0.5 mmol/L) (Spectrozyme; American Diagnostica) to 200 μL of conditioned medium and monitored at 405 nm. For direct assessment of SLPI on plasmin activity, plasmin was incubated with 50 μL of buffer (50 mmol/L Tris + 0.01% Tween 80), 50 μL of SLPI (10 μg/mL) or aprotinin [10 kallikrein inhibitor units (KIU)/mL; American Diagnostica], a plasmin inhibitor, for 15 minutes at 37°C and added to plasmin-specific amidolytic substrate (Spectrozyme PL) in a 96-well format. Increase in absorbance at 405 nm was directly proportional to the amount of plasmin generated. Fibrin gels were prepared in 12-well plates (Corning Costar; Corning Life Sciences, Kennebunk, ME) as described.37Paul-Brent P.A. Cattley T.N. Myers S.P. Brooks L. Cheras P.A. A rapid microtitre plate screening method for in vitro assessment of fibrinolysis: a preliminary report.Blood Coagul Fibrinolysis. 2004; 15: 273-278Crossref PubMed Scopus (2) Google Scholar Briefly, bovine plasma fibrinogen (Sigma-Aldrich) was added to saline barbital buffer (0.05 mol/L sodium barbital, 0.093 mol/L NaCl, 1.66 mmol/L CaCl2, 0.69 mmol/L MgCl2, ionic strength 0.15, pH adjusted to 7.75) at a final concentration of 0.1% w/v and dispensed at 400 μL/well. The fibrinogen solution was clotted with 18 μL (20 NIH units/mL) bovine thrombin solution (Sigma) for 60 minutes. A 30-μL test sample in gelatin barbital buffer (containing 0.25% gelatin) was added to individual wells at 37°C for 17 hours37Paul-Brent P.A. Cattley T.N. Myers S.P. Brooks L. Cheras P.A. A rapid microtitre plate screening method for in vitro assessment of fibrinolysis: a preliminary report.Blood Coagul Fibrinolysis. 2004; 15: 273-278Crossref PubMed Scopus (2) Google Scholar in parallel with plasmin (0.0498 ng) and tPA (0.03 μg; Calbiochem). Aprotinin (1unit/mL) or PAI-1 (0.67 units/mL) was added to some wells at the onset of the experiment. For cell-dependent fibrinolysis, cells were pelleted, resuspended in gelatin barbital buffer, and 50 μL of cells/buffer were added to fibrin plates for 1 to 3 days. Wells were stained with Coomassie Blue, and replicate lysis diameters were quantified. Annexin A2 small-interfering (siRNA) was synthesized based on the sequence r(5′-CGGUGAUUUUGGGCCUAUUUU-3′)/r(5′-AAUAGGCCCAAAUCACCGUC-3′) and an unrelated control siRNA from Qiagen (Valencia, CA).23Ma G. Greenwell-Wild T. Lei K. Jin W. Swisher J. Hardegen N. Wild C.T. Wahl S.M. Secretory leukocyte protease inhibitor binds to annexin II, a cofactor for macrophage HIV-1 infection.J Exp Med. 2004; 200: 1337-1346Crossref PubMed Scopus (172) Google Scholar siRNA was transfected into monocytes using Amaxa Human Dendritic Cell Nucleofector kit (Amaxa Biosystems, Köln, Germany) as described.23Ma G. Greenwell-Wild T. Lei K. Jin W. Swisher J. Hardegen N. Wild C.T. Wahl S.M. Secretory leukocyte protease inhibitor binds to annexin II, a cofactor for macrophage HIV-1 infection.J Exp Med. 2004; 200: 1337-1346Crossref PubMed Scopus (172) Google Scholar The cells were incubated 6 days before analysis of annexin protein using Western blot and assay for fibrinolytic activity. Adherent macrophages (6 × 106/well in a six-well plate) were treated with adenovirus vector (1.46 × 1012 viral particles/mL, 4 μL added to 1 mL of 10% FCS in DMEM), adenovirus-GFP, or adenovirus-SLPI (7.2 × 1011 viral particles/mL, 8.3 μL in 1 mL). After 24 hours, medium was removed and replaced with 10% DMEM until days 3 to 7, when supernatants were tested for SLPI by ELISA (R&D Systems). Cells were detached, washed, and used in plasmin generation or fibrinolytic assays. HN12 cells were cultured on coverslips in 24-well plates until 75% to 90% confluent, washed, and then fixed with 4% paraformaldehyde before incubation with 100 mmol/L glycine for 25 minutes. Cells were washed, permeabilized with ice-cold 100% methanol, and blocked with 2% donkey serum in PBS for 10 minutes before monoclonal anti-annexin A2, rabbit polyclonal anti-SLPI (Santa Cruz Biotechnology), and control antibodies were applied to cells at 4°C overnight. Fluorophore-labeled secondary antibodies (1 μL/200 μL of buffer; Invitrogen) were added for 1 hour at room temperature; cells were then washed and incubated with DAPI (Sigma-Aldrich) for 10 minutes. Slides were visualized microscopically (Zeiss Axioplan 2 Imaging; Carl Zeiss, Oberkochen, Germany) with filters for fluorescein isothiocyanate, tetramethyl rhodamine, and DAPI/HOECHST, and recorded with iVision-Mac Image Acquisition and Analysis software (BioVision Technologies, Exton, PA). Macrophages were rinsed with PBS, incubated in DMEM containing biotinylated rhSLPI,23Ma G. Greenwell-Wild T. Lei K. Jin W. Swisher J. Hardegen N. Wild C.T. Wahl S.M. Secretory leukocyte prot
