A human bronchial xenograft model was used to characterize the molecular basis for the previously described defect in bacterial killing that is present in the cystic fibrosis (CF) lung. Airway surface fluid from CF grafts contained abnormally high NaCl and failed to kill bacteria, defects that were corrected with adenoviral vectors. A full-length clone for the only known human beta-defensin (i.e., hBD-1) was isolated. This gene is expressed throughout the respiratory epithelia of non-CF and CF lungs, and its protein product shows salt-dependent antimicrobial activity to P. aeruginosa. Antisense oligonucleotides to hBD-1 ablated the antimicrobial activity in airway surface fluid from non-CF grafts. These data suggest that hBD-1 plays an important role in innate immunity that is compromised in CF by its salt-dependent inactivation.

We have previously shown that antimicrobial peptides like defensins have the capacity to mobilize leukocytes in host defense. LL-37 is the cleaved antimicrobial 37-residue, COOH-terminal peptide of hCAP18 (human cationic antimicrobial protein with a molecular size of 18 kD), the only identified member in humans of a family of proteins called cathelicidins. LL-37/hCAP18 is produced by neutrophils and various epithelial cells. Here we report that LL-37 is chemotactic for, and can induce Ca2+ mobilization in, human monocytes and formyl peptide receptor–like 1 (FPRL1)-transfected human embryonic kidney 293 cells. LL-37–induced Ca2+ mobilization in monocytes can also be cross-desensitized by an FPRL1-specific agonist. Furthermore, LL-37 is also chemotactic for human neutrophils and T lymphocytes that are known to express FPRL1. Our results suggest that, in addition to its microbicidal activity, LL-37 may contribute to innate and adaptive immunity by recruiting neutrophils, monocytes, and T cells to sites of microbial invasion by interacting with FPRL1.

The stratified epithelia of the oral cavity are continually exposed to bacterial challenge that is initially resisted by innate epithelial factors and by the recruitment of neutrophils. Antimicrobial peptides from phagocytes and epithelia contribute to this antimicrobial barrier. Using antibodies and in situ hybridization, we explored antimicrobial peptide expression in the varied epithelia of the periodontium and in cultured gingival epithelial cells. In gingival tissue, mRNA for the beta-defensins, human beta-defensin 1 (hBD-1) and human beta-defensin 2 (hBD-2) was predominately localized in suprabasal stratified epithelium and the peptides were detected in upper epithelial layers consistent with the formation of the stratified epithelial barrier. In cultured epithelial cells, both hBD-1 and -2 peptides were detected only in differentiating, involucrin-positive epithelial cells, although hBD-2 required stimulation by proinflammatory mediators or bacterial products for expression. Beta-defensins were not detected in junctional epithelium (JE) that serves as the attachment to the tooth surface. In contrast, alpha-defensins and cathelicidin family member LL-37 were detected in polymorphonuclear neutrophils (PMNs) that migrate through the JE, a localization that persists during inflammation, when the JE and surrounding tissue are highly infiltrated with PMNs. Thus, the undifferentiated JE contains exogenously expressed alpha-defensins and LL-37, and the stratified epithelium contains endogenously expressed beta-defensins. These findings show that defensins and other antimicrobial peptides are localized in specific sites in the gingiva, are synthesized in different cell types, and are likely to serve different roles in various regions of the periodontium.

Vascular complications are an important cause of morbidity and mortality in patients with diabetes. The extent of vascular complications has been linked statistically to enhanced adherence ofdiabetic erythrocytes to endothelial cells (ECs) and to the accumulation of a class of glycated proteins termed advanced glycation end products (AGEs). We Nonenzymatic glycation ofproteins, such as hemoglobin, has been shown to provide a useful index for management of patients with diabetes (1). The ultimate result of the nonenzymatic glycation and oxidation of proteins is formation of advanced glycation end products (AGEs), whose presence in plasma and tissues has been linked to the development of complications in diabetics (2-5). The cellular interactions of AGEs are mediated by receptors/cell surface binding proteins identified on endothelial cells (ECs) and mononuclear phagocytes (MPs), engagement of which leads to perturbation of cellular functions (3,(6)(7)(8). Our studies have characterized an integral membrane protein, receptor for AGE (RAGE), a newly discovered member of the immunoglobulin superfamily, which has a central role in mediating the interactions of AGEs with cellular surfaces (7-9).We previously showed that erythrocytes from diabetic patients exhibited enhanced binding to cultured endothelium (10). We hypothesized that, dependent on the duration of exposure of erythrocytes to plasma hyperglycemia, AGE modification oferythrocyte surface membrane proteins could occur, allowing them to bind and thereby to modulate properties of RAGE-expressing vessel wall cells. Our studies demonstrate that the molecular basis of the increased adherence of diabetic erythrocytes results largely from AGEs on the erythrocyte surface interacting with EC RAGE. This results in the induction of oxidant stress in the endothelium, potentially modulating expression of a spectrum ofgenes that could contribute to the pathogenesis of vascular complications.MATERIALS AND METHODS Subjects. The group of patients (n = 18 each for the normal and diabetic subjects) was comparable in age, duration of diabetes, fasting blood glucose, and hemoglobin Alc levels.Erythrocytes from two patients homozygous for sickle cell disease were also studied.Erythrocyte Adhesion Assay. Cultured human umbilical vein ECs were prepared and assayed as described (10-12).The specific activity of 51Cr-labeled erythrocytes for normal and diabetic erythrocytes was 3750 ± 260 and 3820 ± 253 cpm per mg of hemoglobin, respectively. The adhesion ratio (AR) was calculated as follows: AR = (cpm of diabetic erythrocytes)/(cpm of normal erythrocytes). An AR value of 1 represents the adhesion observed with normal erythrocytes. Where indicated, either erythrocytes or ECs were preincubated with soluble RAGE (sRAGE) or antibodies and EC nuclear extracts were prepared (13).Preparation of AGE-Modified Proteins, AGE Binding Proteins, and Antisera. AGE albumin was prepared and characterized as described (3,7,8). AGE binding proteins were purified from bovine lung (7) and cons...

ErbB oncogenes drive the progression of several human cancers. Our study shows that in human carcinoma (A431) and glioma (U373) cells, the oncogenic forms of epidermal growth factor receptor (EGFR; including EGFRvIII) trigger the up-regulation of tissue factor (TF), the transmembrane protein responsible for initiating blood coagulation and signaling through interaction with coagulation factor VIIa. We show that A431 cancer cells in culture exhibit a uniform TF expression profile; however, these same cells in vivo exhibit a heterogeneous TF expression and show signs of E-cadherin inactivation, which is coupled with multilineage (epithelial and mesenchymal) differentiation. Blockade of E-cadherin in vitro, leads to the acquisition of spindle morphology and de novo expression of vimentin, features consistent with epithelial-to-mesenchymal transition. These changes were associated with an increase in EGFR-dependent TF expression, and with enhanced stimulation of vascular endothelial growth factor production, particularly following cancer cell treatment with coagulation factor VIIa. In vivo, cells undergoing epithelial-to-mesenchymal transition exhibited an increased metastatic potential. Furthermore, injections of the TF-blocking antibody (CNTO 859) delayed the initiation of A431 tumors in immunodeficient mice, and reduced tumor growth, vascularization, and vascular endothelial growth factor expression. Collectively, our data suggest that TF is regulated by both oncogenic and differentiation pathways, and that it functions in tumor initiation, tumor growth, angiogenesis, and metastasis. Thus, TF could serve as a therapeutic target in EGFR-dependent malignancies.