ABSTRACT
Chemokines are important in inflammation and in carcinogenesis. We hypothesized that besides oro-laryngeal cancer, oral inflammatory states, such as periodontitis, may also influence the chemokine profile of oral fluid. The aim of this study was to characterize the chemokine isoforms in the oral fluid of patients with periodontitis and in the oral fluid of patients with head and neck cancer. Using enzyme-linked immunosorbent assays (ELISA), it was found that the concentrations of CXCL8, CXCL10, and CCL14 were significantly elevated in the oral fluids of the cancer patients. However, periodontitis did not significantly alter the chemokine levels in oral fluid. Identification of chemokine isoforms by a proteomic approach using a newly developed three-step purification procedure was applied on the oral fluid of head and neck cancer and periodontitis patients and on the conditioned medium from carcinoma cells. Carcinoma cells produced predominantly intact CXCL1, CXCL2, CXCL8, and CCL2, whereas CXCL8 also appeared in a truncated, more active, form. Unfortunately, the chemokine concentrations in oral fluids were too low to allow full biochemical identification of the modified isoforms. However, the chemokine profile of head and neck cancer significantly changed after therapy, indicating that it is a useful parameter in clinical practice.
Subject(s)
Carcinoma, Squamous Cell/immunology , Chemokines/analysis , Laryngeal Neoplasms/immunology , Mouth Neoplasms/immunology , Saliva/immunology , Salivary Proteins and Peptides/analysis , Adult , Aged , Aged, 80 and over , Carcinoma/immunology , Carcinoma, Squamous Cell/therapy , Chemokine CCL2/analysis , Chemokine CXCL1/analysis , Chemokine CXCL10/analysis , Chemokine CXCL2/analysis , Chemokines, CC/analysis , Chemokines, CXC/analysis , Chronic Periodontitis/immunology , Chronic Periodontitis/therapy , Culture Media, Conditioned , Female , HeLa Cells , Humans , Laryngeal Neoplasms/therapy , Male , Middle Aged , Mouth Neoplasms/therapy , Protein Isoforms/analysis , Protein Processing, Post-Translational , ProteomeABSTRACT
Interactions between chemokines and enzymes are vital in immunoregulation. Structural protein citrullination by peptidylarginine deiminase (PAD) has been associated with autoimmunity. In this report, we identified a novel naturally occurring posttranslational modification of chemokines, that is, the deimination of arginine at position 5 into citrulline of CXC chemokine ligand 10 (CXCL10) by rabbit PAD and human PAD2. Citrullination reduced (>/= 10-fold) the chemoattracting and signaling capacity of CXCL10 for CXC chemokine receptor 3 (CXCR3) transfectants; however, it did not affect CXCR3 binding. On T lymphocytes, though, citrullinated CXCL10 remained active but was again weaker than authentic CXCL10. PAD was also able to convert CXCL11, causing an impairment of CXCR3 signaling and T-cell activation, though less pronounced than for CXCL10. Similarly, receptor binding properties of CXCL11 were not altered by citrullination. However, deimination decreased heparin binding properties of both CXCL10 and CXCL11. Overall, chemokines are the first immune modulators reported of being functionally modified by citrullination. These data provide new structure-function dimensions for chemokines in leukocyte mobilization, disclosing an anti-inflammatory role for PAD. Additionally because citrullination has severe consequences for chemokine biology, this invites to reassess the involvement and impact of PAD and citrullinated peptides in inflammation, autoimmunity, and hematologic disorders.
Subject(s)
Chemokine CXCL10/immunology , Chemokine CXCL11/immunology , Citrulline/metabolism , Hydrolases/metabolism , Protein Processing, Post-Translational , Amino Acid Sequence , Animals , Cell Line , Chemokine CXCL10/chemistry , Chemokine CXCL10/isolation & purification , Chemokine CXCL10/pharmacology , Chemokine CXCL11/pharmacology , Glycosaminoglycans/metabolism , Heparin/metabolism , Humans , Lymphocyte Activation/drug effects , Molecular Sequence Data , Phytohemagglutinins/pharmacology , Protein Binding/drug effects , Protein Isoforms/metabolism , Protein Processing, Post-Translational/drug effects , Protein-Arginine Deiminase Type 2 , Protein-Arginine Deiminases , Rabbits , Receptors, CXCR/metabolism , Receptors, CXCR3/metabolism , T-Lymphocytes/cytology , T-Lymphocytes/drug effects , TransfectionABSTRACT
Human granulocyte chemotactic protein-2 (GCP-2)/CXCL6 is a CXC chemokine that functionally uses both of the IL-8/CXCL8 receptors to chemoattract neutrophils but that is structurally most related to epithelial cell-derived neutrophil attractant-78 (ENA-78)/CXCL5. This study provides the first evidence that GCP-2 protein is, compared with IL-8, weakly produced by some sarcoma, but less by carcinoma cells, and is tightly regulated in normal mesenchymal cells. IL-1beta was the predominant GCP-2 inducer in fibroblasts, chondrocytes, and endothelial cells, whereas IL-8 was equally well up-regulated in these cells by TNF-alpha, measles virus, or double-stranded RNA (dsRNA). In contrast, lipopolysaccharide (LPS) was a relatively better stimulus for GCP-2 versus IL-8 in fibroblasts. IFN-gamma down-regulated the GCP-2 production in fibroblasts induced by IL-1beta, TNF-alpha, LPS, or dsRNA. The kinetics of GCP-2 induction by IL-1beta, LPS, or dsRNA in fibroblasts differed from those of IL-8. Freshly isolated peripheral blood mononuclear leukocytes, which are a good source of IL-8 and ENA-78, failed to produce GCP-2. However, lung macrophages and blood monocyte-derived macrophages produced GCP-2 in response to LPS. Quantitatively, secretion of GCP-2 always remained inferior to that of IL-8, despite the fact that the ELISA recognized all posttranslationally modified GCP-2 isoforms. The expression of GCP-2 was confirmed in vivo by immunohistochemistry. The patterns of producer cell types, inducers and kinetics and the quantities of GCP-2 produced, suggest a unique role for GCP-2 in physiologic and pathologic processes.
