Proliferatory defect of invariant population and accumulation of non-invariant CD1d-restricted natural killer T cells in the joints of RA patients.

OBJECTIVES: While numerical and functional defects of invariant NKT cells have been demonstrated in rheumatoid arthritis (RA), the detailed characterization of proliferative and secretory responses following CD1d-mediated presentation is lacking; the presence of non-invariant populations has never been assessed in human autoimmunity. We have evaluated both invariant and non-invariant populations in the blood and synovial fluid from patients to assess feasibility of NKT cell-directed manipulations in RA. METHODS: NKT cell populations were quantified by anti-CD4/anti-Vα24 staining and/or CD1d tetramers. Proliferation was measured in cultures of mononuclear cells following stimulations with αGalCer and cytokine secretion determined by multi-bead assay. RESULTS: We have confirmed a proliferative defect of iNKT cells in both peripheral blood and synovial fluid from RA patients, but no changes in baseline frequencies. Moreover, we have detected an enlargement of non-invariant cell pool in synovial fluid samples. In addition, we noted an evident Th2 shift following exposure to αGalCer and pronounced IL-6 secretion. CONCLUSIONS: While RA patients suffer from defective proliferative responses of invariant NKT cells, non-invariant cells accumulate at the site of inflammation. While stimulation with αGalCer results in reduced TNF-α and increased suppressive IL-10, abundantly produced IL-6 could potentially contribute to the induction of Th17 cells in the joints.

The mucosal immune response to laryngopharyngeal reflux.

RATIONALE: Laryngopharyngeal reflux (LPR) affects up to 20% of Western populations. Although individual morbidity is usually moderate, treatment costs are high and there are associations with other diseases, including laryngeal cancer. To date, there have been no studies of the mucosal immune response to this common inflammatory disease. OBJECTIVES: To determine the mucosal immune response to LPR. METHODS: We performed a prospective immunologic study of laryngeal biopsies from patients with LPR and control subjects (n = 12 and 11, respectively), and of primary laryngeal epithelial cells in vitro. MEASUREMENTS AND MAIN RESULTS: Quantitative multiple-color immunofluorescence, using antibodies for lymphocytes (CD4, CD8, CD3, CD79, CD161), granulocytes (CD68, EMBP), monocytic cells (CD68, major histocompatibility complex [MHC] class II), and classical and nonclassical MHC (I, II, beta(2)-microglobulin, CD1d). Univariate and multivariate analysis and colocalization measurements were applied. There was an increase in percentage area of mucosal CD8(+) cells in the epithelium (P < 0.005), whereas other leukocyte and granulocyte antigens were unchanged. Although epithelial MHC class I and II expression was unchanged by reflux, expression of the nonclassical MHC molecule CD1d increased (P < 0.05, luminal layers). In vitro, laryngeal epithelial cells constitutively expressed CD1d. CD1d and MHC I expression were inversely related in all subjects, in a pattern which appears to be unique to the upper airway. Colocalization of natural killer T (NKT) cells with CD1d increased in patients (P < 0.01). CONCLUSIONS: These data indicate a role for the CD1d-NKT cell axis in response to LPR in humans. This represents a useful target for novel diagnostics and treatments in this common condition.

Immunologic response of the laryngeal mucosa to extraesophageal reflux.

OBJECTIVES: Extraesophageal reflux is common. The treatment costs are high, and there are associations with other diseases, including laryngeal cancer. Our studies of the mucosal immune response to this common inflammatory disease suggest an important role for the nonclassic antigen-presenting molecule CD1d in the response to inflammation. This study was performed to further explore the relationship between the CD1d-NKT cell-iGb3 axis and reflux. METHODS: We carried out a prospective study of laryngeal biopsies from 12 patients with laryngopharyngeal reflux and 11 controls. Quantitative multiple-color immunofluorescence using antibodies for lymphocytes (CD3, CD161) and classic and nonclassic major histocompatibility complex (I, II, beta2m, CD1d) was performed, and univariate and multivariate analysis and co-localization measurements were applied. RESULTS: Epithelial major histocompatibility complex class I and II expression was unchanged by reflux, but expression of CD1d increased (p < 0.05; luminal layers) and confidence intervals diminished in the reflux group. Co-localization of NKT cells with CD1d increased in patients (p < 0.01); iGb3 exhibited strong expression throughout all layers of the laryngeal epithelium. CONCLUSIONS: These data indicate a role for the CD1d-NKT cell-iGb3 axis in response to extraesophageal reflux in humans. This represents a useful target for novel diagnostics and treatments for this common condition.

Optimal method for isolation of human peritoneal mesothelial cells from clinical samples of omentum.

INTRODUCTION: Human peritoneal mesothelial cells (HPMC) are a valuable research tool for understanding the molecular biology of several pathologies, in both monolayer and three dimensional models. We compared different methods of HPMC isolation and assessed their outcome as well as fibroblast contamination, a common problem encountered during isolation. METHODS: 1-3cm(3) samples of omentum were collected from 40 consenting patients undergoing elective gastrointestinal surgery. A total of 11 samples were incubated in 0.05% trypsin solution for 20 minutes at 37 degrees C (group A) and 29 in 0.25% trypsin (15 samples for 10 minutes (group B) and 14 for 20 minutes (group C)). Following digestion cells were re-suspended and cultured in supplemented Ham's F-12 medium containing 10% foetal calf serum (FCS), penicillin-streptomycin, glutamine, insulin, transferrin and hydrocortisone. Positive outcomes were absence of fibroblast contamination and satisfactory HPMC growth to confluence in a characteristic cobblestone pattern. Cytokeratins 5, 8, 18, Vimentin, Ber-Ep4 and Factor VIII were used to characterise HPMC and fibroblasts by immunohistochemistry. RESULTS: None of the 11 samples in group A yielded HPMC. 14 of 29 samples digested with 0.25% trypsin yielded HPMC: 10 of 14 yielded HPMC in group C versus four of 15 samples in group B (p = 0.02). Fibroblast contamination occurred in eight samples in group B versus three in group C. CONCLUSION: Optimal results are achieved with a 20 minute digestion in 0.25% trypsin. Fibroblast contamination could not be avoided completely. Other factors may minimise fibroblast contamination such as minimal tissue manipulation and early collection during surgery.

Inflammatory gene expression patterns revealed by DNA microarray analysis in TNF-alpha-treated SGBS human adipocytes.

We report here the use of human inflammation arrays to study the inflammatory gene expression profile of TNF-alpha- treated human SGBS adipocytes. Human preadipocytes (SGBS) were induced to differentiate in primary culture, and adipocyte differentiation was confirmed, using Oil Red O staining. We treated the differentiated adipocytes with TNF-alpha, and RNA from differentiated adipocytes with or without TNF-alpha treatment was hybridized to MWG human inflammation arrays to compare expression profiles. Eleven genes were up- or down-regulated in TNF-alpha-treated adipocytes. As revealed by array analysis, among 6 up-regulated genes, only eotaxin-1, monocyte chemoattractant protein-1 (MCP-1), and vascular cell adhesion molecule 1 isoform a precursor (VCAM1) were confirmed by real-time polymerase chain reaction (PCR). Similarly, among 5 down-regulated genes, only IL-1 family member 5 (IL1F5), a disintegrin and metalloprotease with thrombospondin motifs-1 preproprotein (ADAMTS1), fibronectin 1 isoform 1 preprotein (FN1), and matrix metalloproteinase 15 preprotein (MMP15) were confirmed by real-time PCR. There was a substantial increase (50-fold) in eotaxin-1 in response to TNF-alpha. Taken together, we have identified several inflammatory molecules expressed in SGBS adipocytes and discovered molecular factors explaining the relationship between obesity and atherosclerosis, focusing on inflammatory cytokines expressed in the TNF-alpha-treated SGBS cells. Further investigation into the role of these up- or down-regulated cytokine genes during the pathological processes leading to the development of atherosclerosis is warranted.

Do NK cells regulate human autoimmunity?

Natural killer cells have been shown to regulate autoimmune responses under experimental conditions in animals. However, a similar role for human NK cells has not been investigated, although NK cells constitute a significant fraction of the infiltrating cells in a range of autoimmune diseases. This review investigates the evidence, both theoretical and experimental, for the involvement of these cells in human immunopathology.