Deanne M. R. Lathers

Human squamous cell carcinomas of the head and neck chemoattract immune suppressive CD34+ progenitor cells

CD34(+) progenitor cells have previously been shown to be mobilized in patients with squamous cell carcinoma of the head and neck (HNSCC). The present study showed that these CD34(+) cells inhibit the capacity of intratumoral lymphoid cells to become activated in response to stimulation through the TCR/CD3 complex. The mechanisms that could lead to the accumulation of CD34(+) cells within the tumor tissue were assessed. This was accomplished through in vitro studies that determined if HNSCC produce soluble factors that chemoattract CD34(+) cells. The migration of cord blood CD34(+) cells, which were used as a readily available source of progenitor cells, was stimulated by products derived from HNSCC explants and primary HNSCC cultures. This stimulated migration was due to chemotaxis because it was dependent on an increasing gradient of HNSCC-derived products. CD34(+) cells that were isolated from the peripheral blood of HNSCC patients were similarly chemoattracted to the HNSCC-derived products. The majority of the chemotactic activity produced by HNSCC could be attributed to vascular endothelial cell growth factor (VEGF). These studies indicate that HNSCC can chemoattract immune inhibitory CD34(+) progenitor cells through their production of VEGF.

Antigen-specific IgE in sinus mucosa of allergic fungal rhinosinusitis patients.

Background Local tissue production of antigen-specific immunoglobulin E (IgE) has been shown in patients with allergic rhinitis and in patients with chronic rhinosinusitis (CRS) with nasal polyps. In allergic fungal rhinosinusitis (AFRS), specific IgE has been established in nasal lavage fluid and eosinophilic mucin. In this study, local production of antigen-specific IgE within sinus mucosa of AFRS patients was evaluated. Methods Sinus mucosa homogenates from 11 AFRS patients, 8 patients with CRS without nasal polyps (CRSsNP), and 9 nonrhinosinusitis control patients were assessed for IgE localization by immunohistochemistry. AFRS and control tissue homogenates were also evaluated for antigen-specific IgE to 14 common antigens by ImmunoCAP testing (Phadia AB, Portage, MI). Results There was a significant increase in IgE staining in AFRS sinus epithelium and subepithelium compared with controls and with patients with CRSsNP (p ≤ 0.012 for all group differences). AFRS patients showed increased IgE staining in the subepithelium when compared with epithelium (p < 0.001). AFRS sinus tissue had significantly more IgE measured by ImmunoCAP when compared with control sinus tissue for 7 of 14 specific antigens (p < 0.05) and for total IgE (p = 0.004). Antigens with a significant difference on ImmunoCAP included Cladosporium, Aspergillus, Timothy grass, red maple, cockroach, ragweed, and cocklebur. Conclusion AFRS patients showed significantly more IgE in sinus mucosa tissue specimens, with increased IgE in subepithelial sites when compared with epithelium. The increased expression of antigen-specific IgE is not limited to fungal antigens. These findings support the role of type I hypersensitivity and local manifestations of allergy in AFRS patients.

Immunolocalization of surfactant protein A and D in sinonasal mucosa.

Background Surfactant-associated proteins (SP) A and D are in the family of collectin proteins that play an integral part in the innate defense system. SP-A and SP-D expression and function are altered in a variety of inflammatory and infectious diseases of the lungs, such as asthma, allergies, and cystic fibrosis. Our prior studies are the first to identify the presence of these proteins in the human sinonasal cavity. The objective of this study was to immunolocalize SP-A and SP-D in human sinonasal tissue. Methods Sinonasal mucosal biopsies were performed in patients with various forms of chronic hyperplastic rhinosinusitis with nasal polyposis and nondiseased mucosa from patients undergoing transsphenoidal hypophysectomy. (n = 10) Immunolocalization of surfactant proteins was performed with antibodies to SP-A and SP-D using immunoperoxidase staining technique. Isotype-negative controls were performed on all specimens. Results Analyses of mucosal biopsy specimens from human sinonasal tissue reveals staining within respiratory and intermediate (metaplastic)-type surface epithelium. In addition, staining was intense in the submucosal ductal epithelium of the seromucinous glands. These properties appear to be consistent regardless of disease state and location within the sinuses. Conclusion This is the first study to immunolocalize SP-A and SP-D in sinonasal human mucosa. These are secreted proteins that are intricately involved in innate immunity in the lungs. Their secretion in the upper airway indicates that future studies may allow manipulation of these proteins and development of novel treatments for sinonasal pathology.

Local production of antigen-specific IgE in different anatomic subsites of allergic fungal rhinosinusitis patients

Objective: Local production of antigen-specific IgE in allergic fungal rhinosinusitis (AFRS) is likely integral to the expression of allergy. This study examines if there are anatomic variations in local IgE expression or if variations among fungal and nonfungal IgE exist. Study Design: Cross-sectional study. Setting: Tertiary medical center. Subjects and Methods: Specimens from 11 AFRS, 8 chronic rhinosinusitis without nasal polyps (CRSsNP), and 9 control patients underwent immunohistochemical localization for IgE and evaluation for antigen-specific IgE by ImmunoCAP testing. Results: Inferior turbinate (IT) epithelium had greater IgE staining in AFRS than control (P = 0.013) and CRSsNP (P = 0.002). A significant difference was also found at the IT subepithelial level for AFRS compared with controls (P = 0.001) and CRSsNP (P < 0.001). Within AFRS, IgE staining was increased in the subepithelium compared to epithelium (P = 0.003). ImmunoCAP analysis on IT tissue from AFRS and controls demonstrated increased antigen-specific IgE for 5 of 14 antigens (P < 0.05) and total IgE (P < 0.001). There were no significant anatomic differences between IT and sinus IgE staining. Conclusion: More fungal and nonfungal IgE is expressed in IT and sinus tissues of AFRS patients, as compared with control and CRSsNP patients.

Tumors skew endothelial cells to disrupt NK cell, T-cell and macrophage functions.

IntroductionPatients and mice with solid tumors, such as Lewis lung carcinoma (LLC), have defects in functions of immune effector cells. Endothelial cells, a component of the tumor vasculature, are potential regulators of immune cell functions. Therefore, these studies examined the impact of exposure to LLC tumor on the ability of endothelial cells to modulate immune cell functions.Materials and methodsEndothelial cells were pre-treated with LLC tumor-conditioned medium (EndoT-sup) for 24 h. Control endothelial cells that were exposed to medium (EndoMedia) or epithelial cell-conditioned medium (EndoEpi-sup). After the initial 24 h incubation, endothelial cells were washed and fresh media was added. Cells were allowed to incubate for an additional 24 h. Supernatants from EndoMedia, EndoEpi-sup or EndoT-sup were collected and assayed for immune modulatory products and for immune modulatory activity.ResultsSupernatant from EndoT-sup contained increased levels of PGE2, IL-6 and VEGF as compared to EndoMedia and EndoEpi-sup controls. NK cell activity, as measured by TNF-α and IFN-γ secretion, was increased following exposure to media conditioned by EndoMedia and EndoEpi-sup. Exposure of NK cells to supernatants of EndoT-sup, also increases TNF-α and IFN-γ secretion, but to a lesser extent than by EndoMedia and EndoEpi-sup. Examination of macrophage functions demonstrated that supernatant from EndoT-sup decreased microbead phagocytosis and increased production of the immune suppressive mediators, IL-10 and PGE2. Lastly, T-cell responses to stimulation with anti-CD3 in the presence of supernatants from EndoT-sup were examined. IFN-γ production by CD8+ T-cells was reduced after exposure to EndoT-sup-conditioned medium, as compared to cells treatments with medium or control conditioned medium. Production of IFN-γ by CD4+ T-cells exposed to EndoT-sup was not altered.ConclusionsTaken together, these studies demonstrate that tumors skew endothelial cells to disrupt NK cell, T-cell and macrophages functions, and represents a novel mechanism of tumor-induced immune suppression.

Immunolocalization of dendritic cells and pattern recognition receptors in chronic rhinosinusitis

Background Dendritic cell (DC) activation and antigen presentation to T cells are critical to innate and adaptive immunity. Toll-like receptors (TLRs) are known to bind pathogen-associated molecular patterns in addition to sinonasally secreted surfactant proteins (SP) such as SP-A and SP-D. TLR binding is known to activate DCs. Based on these observations, we sought to establish the presence, in sinonasal mucosa, of DC and the pattern recognition receptors (PRRs), CD14, TLR2, and TLR4. Methods Sinonasal biopsy specimens were taken from patients with eosinophilic nonatopic nasal polyposis (n = 4), allergic fungal sinusitis (n = 1), and nondiseased patients undergoing cerebrospinal fluid leak repair or pituitary tumor resection (n = 2). Tissue samples were stained immunohistochemically for PRR (CD14, TLR2, and TLR4), mature DC marker (CD208), iDC marker (CD209), or isotype controls. Results Immature and mature DC were immunolocalized to the subepithelial stroma and ciliated epithelial surface, respectively. Diffuse staining of CD14 was observed throughout the stroma with additional staining in the ciliated epithelium. The TLR markers showed no staining in the ciliated epithelium. TLR2 primarily localized in stroma immediately deep to the ciliated epithelial surface. TLR4 immunolocalized to submucosal seromucinous gland ductal epithelium. Data from nondiseased patients were mixed, with one patient showing minimal staining of any of the tested cellular markers. Conclusion This study indicates progressive DC activation and emigration of mature antigen-presenting cells from the epithelial surfaces of sinonasal mucosa. The presence of TLR known to bind SP-A and SP-D suggests a link between SP expression and immune response in sinonasal mucosa.

Innate and adaptive mediators in cystic fibrosis and allergic fungal rhinosinusitis.

Background Surfactant-associated proteins (SP) A and D are both innate immunity mediators and produced in normal and diseased sinus mucosa. Cystic fibrosis (CF) is associated with Th1 adaptive inflammation whereas allergic fungal rhinosinusitis (AFRS) is associated with Th2 adaptive inflammation. The purpose of this study is to show and quantify the presence of SP A, SP D, tumor necrosis factor (TNF) alpha, (a Th1 marker), and eotaxin (a Th2 marker) in normal and diseased sinus mucosa. Methods Intraoperative sinus mucosal biopsy specimens from human volunteers were obtained during endoscopic sinus surgery for CF (n = 4), AFRS (n = 10), and normal controls (CTLs; n = 4). Specimens were evaluated for presence and quantity of SP A, SP D, and TNF-alpha using Western blot with semiquantitative immunoblot analysis. Eotaxin was quantified using ELISA immunoassay. Results were standardized and reported as picograms of mediator per microgram of total protein. Results SP A, SP D, and TNF-alpha levels in CF tissue extracts were 2–10 times higher than levels in AFRS tissue (with SP D and TNF-alpha reaching statistical significance) but CF tissue was not significantly higher than CTL tissue. SP A, SP D, and TNF-alpha were not significantly elevated in AFRS. Eotaxin showed elevated levels in CF and AFRS when compared with CTLs (p = 0.03 and 0.003, respectively). Conclusion SP D and TNF-alpha are significantly increased in CF compared with AFRS, suggesting activation of both innate immunity and Th1-mediated inflammation and potential correlation between SPs and downstream adaptive immune responses.

Combination Docetaxel Plus Vitamin D3 as an Immune Therapy in Animals Bearing Squamous Cell Carcinomas

OBJECTIVE: Background tumor growth results in the mobilization of immune inhibitory CD34+ progenitor cells. However, vitamin D3 can differentiate the CD34+ cells into immune stimulatory dendritic cells. This study determined if docetaxel treatment could increase the impact of the vitamin D3 to generate dendritic cells. METHODS: The murine squamous cell carcinoma model, SCC VII/SF, which is often used as a head and neck cancer model, was used to determine the immunological effects of two cycles of docetaxel plus vitamin D3. RESULTS: Vitamin D3 with or without docetaxel was similarly effective in reducing CD34+ cell levels within the spleen, lymph nodes, and tumor. Dendritic cell levels were similarly enhanced in the lymph nodes by vitamin D3 alone or combined with docetaxel. However, the combination treatment caused a prominent increase in intratumoral levels of active T cells, which was not observed by the individual treatments. CONCLUSION: Incorporating docetaxel treatment with vitamin D3 differentiation-inducing treatment enhances intratumoral immune responsiveness.

Autocrine motility-stimulatory pathways of oral premalignant lesion cells

Patients with premalignant oral lesions have varying levels of risk of developing oral squamous cell carcinoma (OSCC), whose aggressiveness requires increased motility. Not known is if and how premalignant oral lesion cells acquire the increased motility characteristic of OSCC. This was addressed by immunohistochemical analysis of banked premalignant lesion tissues and by functional analyses using cultures established from premalignant oral lesions and OSCC. These studies showed premalignant oral lesion cells and OSCC to be more motile than normal keratinocytes. Concomitantly, levels of ceramide were reduced. The activity of the protein phosphatase PP-2A, which restricts motility and which can be activated by ceramide, was also diminished. This was due to IL-10 released from premalignant lesion cells. Treatment with a membrane-permeable ceramide restored PP-2A activity and blocked migration. These studies show an autocrine motility-stimulatory pathway that is mediated in premalignant lesion cells by IL-10 through its reduction of ceramide levels and inhibition of PP-2A activity.

Impact of aging on immune modulation by tumor

Abstract Tumor development and aging can each alter immune competence. The present study aimed to determine the impact of Lewis lung carcinoma (LLC) presence on immune parameters of middle-aged (averaging 6.5 months) versus aged (averaging 21.3 months) mice. An age-associated decline in the CD4+ cell frequency was seen in freshly isolated spleen and lymph node cells, as well as in cultures stimulated with immobilized anti-CD3. This decline was not further exacerbated by tumor presence. What was prominently inhibited by tumor was the capacity of either splenic or lymph node CD4+ cells to become stimulated to express IFN-γ. Spleen and lymph node cultures from aged tumor-bearing mice had the lowest frequency of CD4+IFN-γ+ cells and the least amount of secreted IFN-γ. CD8+ cells were not affected by aging, but tumor presence reduced the induction of CD8+IFN-γ+ cells in lymph node cultures. We previously showed that LLC growth stimulates myelopoiesis, as seen by splenomegaly and the mobilization of immune inhibitory CD34+ progenitor cells. Tumor presence in middle-aged mice reduced spleen cell blastogenesis, which was mediated by CD34+ cells. Aged mice had reduced blastogenesis, and this was further reduced by presence of tumor. However, neither the age-associated immune dysfunction nor the tumor-induced immune suppression in aged mice was due to CD34+ progenitor cells. These studies show how tumor presence can further compromise the immune dysfunction that accompanies aging. In addition, they show that aging impacts on the mechanisms by which tumors inhibit T-cell capabilities, with myelopoiesis-associated CD34+ cells mediating the immune depression of middle-aged tumor-bearers and an independent mechanism being responsible for the immune depression in aged tumor-bearing mice.