Decreased systemic IL-7 and soluble IL-7Rα in multiple sclerosis patients.

Polymorphisms (single-nucleotide polymorphism (SNP)) in the interleukin-7 receptor-α (IL-7Rα)/IL-7 pathway are associated with an increased risk to develop multiple sclerosis (MS). The rs6897932 SNP in the IL-7Rα leads to increased soluble IL-7Rα production. Given the functional interaction between sIL-7Rα, membrane-bound IL-7Rα and IL-7, we assessed IL-7, mIL-7Rα and sIL-7Rα levels in MS patients and healthy controls (HCs). One-hundred and twenty eight MS patients had significantly lower sIL-7Rα levels compared with 73 HCs. The levels of sIL-7Rα increased dose-dependent upon rs6897932 [C] risk allele carriership in both HCs and MS. Next, we hypothesized that lower sIL-7Rα could result in a higher mIL-7Rα to soluble IL-7Rα ratio. Indeed, 52 MS patients had significantly increased mIL-7Rα to sIL-7Rα ratio for both CD4 and CD8 T cells compared with 44 HCs. Given the supposed role of IL-7 in autoimmunity, we determined whether sIL-7Rα influences IL-7 levels. IL-7 levels were significantly decreased in 40 MS patients compared with 40 HCs. In conclusion, MS patients had lower free IL-7 and a higher membrane to soluble IL-7Rα ratio. The soluble IL-7Rα levels correlate with the rs6897932 [C] risk allele carriership. The skew at the IL-7 and IL-7Rα level may influence responsiveness of IL-7Rα(+) cells.

Two different types of sialoadenitis in the NOD- and MRL/lpr mouse models for Sjögren's syndrome: a differential role for dendritic cells in the initiation of sialoadenitis?

Sjögren's syndrome is an autoimmune disease that primarily affects the salivary and lacrimal glands. In these glands, focal lymphocytic infiltrates develop. Little is known about the initiation of this autoimmune disease. Antigen-presenting cells (APC) such as dendritic cells (DC) can play a role in the initiation of autoimmunity. To date, no data on the presence of DC in Sjögren's syndrome are available. Several mouse strains, the nonobese diabetic (NOD) and the MRL/Ipr mouse, can be used as models for Sjögren's syndrome. We compared the development of sialoadenitis in the submandibular glands (SMG) of NOD and MRL/Ipr mice with particular focus on the presence of APC. DC, macrophages, T cells, and B cells in the SMG were studied by means of immunohistochemistry, after which positively stained cells were quantified. NOD-severe combined immunodeficiency (SCID) mice were used to study the presence of APC in the SMG in the absence of lymphocytes. Before lymphocytic infiltration, increased numbers of DC were detected in the SMG of NOD mice compared with those numbers in control mice and MRL/Ipr mice, which suggests that DC play a role in the initiation of sialoadenitis in NOD mice. In the SMG of NOD mice, lymphocytic infiltrates organized in time. In MRL/Ipr mice, however, lymphocytic infiltrates were already organized at the time of appearance. This organization was lost over time. In conclusion, two types of sialoadenitis are described in two mouse models for Sjögren's syndrome. Differences exist with regard to early events that may lead to the development of sialoadenitis and to the composition and organization of inflammatory infiltrates. It is possible that different types of sialoadenitis also exist in humans and that the pathogenetic process in both the early and late phases of the autoimmune reaction differs among patients.

Professional antigen presenting cells in minor salivary glands in Sjögren's syndrome: potential contribution to the histopathological diagnosis?

Sjögren's syndrome is an autoimmune disease in which lymphocytic infiltrates develop in the salivary and lacrimal glands. We have shown that dendritic cells (DC) infiltrate the submandibular gland of the nonobese diabetic (NOD) mouse, a mouse model for Sjögren's syndrome, before lymphocytic infiltration, suggesting that these antigen-presenting cells (APC) may play a role in the initiation of Sjögren's syndrome. In later stages, DC and macrophages also form an important part of the infiltrate of the NOD sialoadenitis. To find out if DC and macrophages form part of the infiltrate in Sjögren's syndrome as well, and to determine whether they may be useful in the histopathological diagnosis of Sjögren's syndrome, we studied their presence in minor salivary glands (MSG) of patients with Sjögren's syndrome and patients with focal lymphocytic sialoadenitis (FLS), but without clinical or serological criteria of Sjögren's syndrome. Immunohistochemistry was applied, followed by semiquantitative analysis. DC and macrophages were present in all MSG; however, there were clear differences in marker expression between Sjögren's syndrome and FLS, on the one hand, and control tissue, on the other hand. CD1a+ DC and RFD9+ macrophages were mainly observed in MSG in which a focal lymphocytic infiltrate was present. In fact, the diffuse presence of single CD1a+ DC and RFD9+ macrophages correlated closely with the presence of a focal lymphocytic infiltrate in the MSG. This indicates that these cells could be of help during the evaluation of a MSG. Because the detection of APC is technically less cumbersome than a focal score, this parameter may perhaps replace the focal score in the histopathological diagnosis of Sjögren's syndrome. This study therefore prompts further investigation focusing on the presence of CD1a+ and RFD9+ cells in the MSG of a large cohort of patients.

Interleukin 4 receptors on human bronchial epithelial cells. An in vivo and in vitro analysis of expression and function.

Asthma is considered a Th2-like disease, characterized by locally increased levels of interleukin (IL) 4. The bronchial epithelium plays an important role in the initiation and perpetuation of inflammatory reactions within the airways. However, little is known about the presence of IL-4 receptors on human bronchial epithelial cells, or the effects of IL-4 on these cells. In this report, definitive evidence of IL-4 receptor expression on human bronchial epithelial cells using several methods is presented. IL-4 receptor expression on human bronchial epithelial cells in vivo was demonstrated using in situ hybridization and immunohistochemistry. No difference in IL-4 receptor protein expression was observed between bronchial biopsies of healthy subjects compared to allergic asthmatics. Cultured human bronchial epithelial cells also expressed IL-4 receptor mRNA and protein (as determined by RT-PCR analysis and flow cytometry, respectively). IL-4 receptor protein expression by bronchial epithelial cells could be increased by stimulation with PMA+calcium ionophore, whereas IL-1beta and IL-6 decreased IL-4 receptor expression. A cyclic AMP analogue and IL-4 had no effect. Finally, it is shown that the IL-4 receptor is functionally active as IL-4 stimulates the release of IL-8, monocyte chemoattractant protein 1, and particularly IL-1 receptor antagonist by human bronchial epithelial cells. It is concluded that human bronchial epithelial cells express IL-4 receptors both in vivo and in vitro. Stimulation of human bronchial epithelial cells by IL-4 may result in the release of both pro- and anti-inflammatory mediators known to be upregulated in asthmatic airways.

Expression of aminopeptidase N and dipeptidyl peptidase IV in the healthy and asthmatic bronchus.

BACKGROUND: Asthma is characterized by reversible airway obstruction, airway hyperresponsiveness, and chronic inflammation of the airways. Since peptides are able to produce many of the pathophysiological features which are characteristic of asthma, peptide-mediated inflammation is thought to play a role in this disease. The effects of peptides are modulated by peptidases, which are able to degrade peptides, mostly resulting in their inactivation. OBJECTIVES: In this study, we investigated the distribution of two peptidases, aminopeptidase N and dipeptidyl peptidase IV, in the human bronchus and determined whether their expression was altered in allergic asthmatics. METHODS: We first determined the distribution of aminopeptidase N and dipeptidyl peptidase IV in the human bronchus using immuno- and enzymehistochemistry and compared this with the distribution of neutral endopeptidase. Secondly, the expression of aminopeptidase N and dipeptidyl peptidase IV was determined in bronchial biopsies of healthy subjects (n = 8) and allergic asthmatics (n = 12). RESULTS: Aminopeptidase N was localized in connective tissue, blood vessels, gland ducts, perichondrium, nerves and leucocytes (mainly mononuclear phagocytes, dendritic cells, and eosinophils). Dipeptidyl peptidase IV was localized in serosal glands, blood vessels, and T cells. Immunohistochemistry and enzymehistochemistry gave similar results. Comparison of the expression of aminopeptidase N and dipeptidyl peptidase IV in bronchial biopsies of healthy controls and atopic asthmatics revealed no significant differences in the lamina propria. In contrast, in the bronchial epithelium of atopic asthmatics an increased number of aminopeptidase N-positive cells could be found. Double-staining identified these cells as L25+ dendritic cells and eosinophils. CONCLUSIONS: We conclude that expression of aminopeptidase N and dipeptidyl peptidase IV is restricted to specific sites within the human bronchus. Furthermore, in the bronchial epithelium of allergic asthmatics an increased number of aminopeptidase N-expressing dendritic cells and eosinophils can be found.

Immunolocalization of interleukin-4 in eosinophils in the bronchial mucosa of atopic asthmatics.

Several studies have shown that human eosinophils can synthesize and release a number of cytokines. The aim of this study was to investigate whether eosinophils contain interleukin (IL)-4 protein. We examined the ultrastructural localization of IL-4 in eosinophils in bronchial mucosal biopsies of 29 nonsmoking atopic asthmatics and 7 controls. No eosinophils were detected in the bronchial mucosa of controls. In the eosinophils (n = 42) of the asthmatics, IL-4 was localized to the electron-dense crystalloid core compartment of 85% of the secondary or specific eosinophil granules (n = 468). Other structures in the eosinophils were unlabeled. Control sections, incubated with an irrelevant primary antibody, were negative. This study demonstrates that pre-formed IL-4 is stored in the secondary eosinophil granules. These results were extended by light microscopic immunodouble-staining for IL-4 protein and eosinophil cationic protein, which showed that a subpopulation of activated eosinophils express IL-4 immunoreactivity in bronchial mucosal biopsies of asthmatics as well as controls. These data indicate that eosinophils may be an important source of IL-4 in allergic inflammation.

Detection of oncogene expression by fluorescent in situ hybridization in combination with immunofluorescent staining of cell surface markers.

To study oncogene expression in heterogeneous cell populations we developed and optimized a non-radioactive in situ hybridization technique using biotinylated single-stranded RNA probes and combined this technique with immunofluorescent staining of cell surface markers. As a model for our studies we used HL60 cells. In these cells we detected c-myc mRNA molecules by in situ hybridization following staining of the pan myeloid cell surface marker CD33, by a monoclonal antibody. Hybrids were detected by streptavidin-FITC and CD33 by a TRITC-conjugated antibody. Controls involved pretreatment with RNAase, hybridization with sense RNA probes and blocking with an excess of unlabeled antisense probes. The integrity of the RNA in the cell was shown by hybridization with the GAPDH antisense probe. Essential for successful double-labeling was the choice of a fixation procedure that was suitable for the in situ hybridization and mild enough not to destroy the cell surface marker staining. This fluorescent in situ hybridization in combination with cell surface marker staining will be useful for studying gene expression in phenotypically well-defined cell populations.