Granzyme M has a critical role in providing innate immune protection in ulcerative colitis.

Inflammatory bowel disease (IBD) is an immunoregulatory disorder, associated with a chronic and inappropriate mucosal immune response to commensal bacteria, underlying disease states such as ulcerative colitis (UC) and Crohn's disease (CD) in humans. Granzyme M (GrzM) is a serine protease expressed by cytotoxic lymphocytes, in particular natural killer (NK) cells. Granzymes are thought to be involved in triggering cell death in eukaryotic target cells; however, some evidence supports their role in inflammation. The role of GrzM in the innate immune response to mucosal inflammation has never been examined. Here, we discover that patients with UC, unlike patients with CD, display high levels of GrzM mRNA expression in the inflamed colon. By taking advantage of well-established models of experimental UC, we revealed that GrzM-deficient mice have greater levels of inflammatory indicators during dextran sulfate sodium (DSS)-induced IBD, including increased weight loss, greater colon length reduction and more severe intestinal histopathology. The absence of GrzM expression also had effects on gut permeability, tissue cytokine/chemokine dynamics, and neutrophil infiltration during disease. These findings demonstrate, for the first time, that GrzM has a critical role during early stages of inflammation in UC, and that in its absence colonic inflammation is enhanced.

Impact of cytokine gene polymorphisms on graft-vs-host disease.

Allogeneic hematopoietic stem cell transplantation (SCT) remains the only available curative therapy for hematological malignancy. It does, however, result in significant morbidity and mortality, predominantly as a consequence of infections, leukemic relapse and graft-vs-host disease (GVHD). While differences in human leukocyte antigen (HLA) molecules between donor and host make a crucial contribution to the alloreactivity driving the donor-antihost response, the cytokine milieu consisting of molecules that both promote and regulate the alloresponse after transplantation is also critical. As such, genetic studies correlating donor and/or host cytokine polymorphisms with disease outcomes have provided useful insight into disease pathogenesis, often confirming effects that have been dissected in animal models of the disease. It is now clear that the polymorphic expression of key cytokines (particularly tumor necrosis factor and interleukin 10) has a demonstrable effect on disease outcome and overall transplant-related mortality. Consideration of the role of genetic polymorphisms in GVHD severity and procedural mortality associated with SCT will lead to improvements in patient outcome such that the addition of non-HLA genetic typing of potential donors will allow optimization of donor selection for a given recipient. This review provides a discussion of the current state of the literature regarding polymorphic expression of the key GVHD cytokines and their capacity to predict clinical disease outcome.

MUC1 epithelial mucin (CD227) is expressed by activated dendritic cells.

The MUC1 mucin (CD227) is a cell surface mucin originally thought to be restricted to epithelial tissues. We report that CD227 is expressed on human blood dendritic cells (DC) and monocyte-derived DC following in vitro activation. Freshly isolated murine splenic DC had very low levels of CD227; however, all DC expressed CD227 following in vitro culture. In the mouse spleen, CD227 was seen on clusters within the red pulp and surrounding the marginal zone in the white pulp. Additionally, we confirm CD227 expression by activated human T cells and show for the first time that the CD227 cytoplasmic domain is tyrosine-phosphorylated in activated T cells and DC and is associated with other phosphoproteins, indicating a role in signaling. The function of CD227 on DC and T cells requires further elucidation.

RelB nuclear translocation regulates B cell MHC molecule, CD40 expression, and antigen-presenting cell function.

Mice with targeted RelB mutations demonstrated an essential role for RelB in immune responses and in myeloid dendritic cell differentiation. Human studies suggested a more global transcriptional role in antigen presentation. Burkitt lymphoma cell lines were used as a model to examine the role of RelB in antigen presentation. After transient transfection of BJAB with RelB, strong nuclear expression of RelB-p50 heterodimers was associated with increased APC function and expression of CD40 and MHC class I. Antisense RelB in DG75 reduced antigen-presenting capacity and CD40-mediated up-regulation of MHC molecules. The data indicate that RelB transcriptional activity directly affects antigen presentation and CD40 synthesis. Stimulation of RelB transcriptional activity may provide a positive feedback loop for facilitating productive APC/T cell interactions.

Differentiated dendritic cells expressing nuclear RelB are predominantly located in rheumatoid synovial tissue perivascular mononuclear cell aggregates.

OBJECTIVE: Differentiated dendritic cells (DC) and other antigen-presenting cells are characterized by the nuclear location of RelB, a member of the nuclear factor kappaB/Rel family. To characterize and enumerate differentiated DC in rheumatoid arthritis (RA) peripheral blood (PB), synovial fluid (SF), and synovial tissue (ST), the expression and location of RelB were examined. METHODS: RelB protein expression and cellular location were determined in RA PB, SF, and ST by flow cytometry and immunohistochemical analysis of purified cells or formalin-fixed tissue. DNA-binding activity of RelB was determined by electrophoretic mobility shift-Western immunoblotting assays. RESULTS: Circulating RA PBDC resembled normal immature PBDC in that they did not express intracellular RelB protein. In RA ST serial sections, cells containing nuclear RelB (nRelB) were enriched in perivascular regions. A mean +/- SD of 84 +/- 10% of these cells were DC. The remaining nRelB+,HLA-DR+ cells comprised B cells and macrophages. Only 3% of sorted SFDC contained nRelB. However, RelB present in the nucleus of these SFDC was capable of binding DNA, and therefore capable of transcriptional activity. CONCLUSION: Circulating DC precursors differentiate and express RelB after entry into rheumatoid ST. Differentiated DC can thus be identified by immunohistochemistry in formalin-fixed ST. Signals for DC maturation may differ between RA ST and SF, resulting in nuclear location of RelB predominantly in ST. This is likely to have functional consequences for the DC in these sites.

Resistance of rheumatoid synovial dendritic cells to the immunosuppressive effects of IL-10.

IL-10 down-regulates the APC function of many dendritic cells (DC), including human peripheral blood (PB) DC. In rheumatoid arthritis (RA), synovial fluid (SF) DC express markers of differentiation and are effective APC despite abundant synovial IL-10. The regulation of DC responsiveness to IL-10 was therefore examined by comparing the effect of IL-10 on normal PB and RA SF DC. Whereas IL-10 down-modulated APC function and MHC class II and B7 expression of PB DC, IL-10 had no such effect on SF DC. Since SF DC have differentiated in vivo in the presence of proinflammatory cytokines, PB DC were cocultured in the presence of IL-10 and either GM-CSF, IL-1beta, TNF-alpha, IL-6, or TGF-beta. GM-CSF, IL-1beta, and TNF-alpha were all able to restore APC function. Whereas the effects of IL-10 on PB DC were shown to be mediated by IL-10R1, neither PB nor RA SF DC constitutively expressed IL-10R1 mRNA or detectable surface protein. In contrast, IL-10R1 protein was demonstrated in PB and SF DC whole cell lysates, suggestive of predominant intracellular localization of the receptor. Thus, DC responsiveness to IL-10 may be regulated through modulation of cell surface IL-10R1 expression or signaling.

Dendritic cells and the pathogenesis of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease in which unknown arthrogenic autoantigen is presented to CD4+ T cells. The strong association of the disease with an epitope within the HLA-DR chain shared between various alleles of HLA-DR4 and DR1 emphasizes the importance of antigen presentation. This immune response predominantly occurs in the synovial tissue and fluid of the joints and autoreactive T cells are readily demonstrable in both the synovial compartment and blood. Circulating dendritic cells (DC) are phenotypically and functionally identical with normal peripheral blood (PB) DC. In the synovial tissue, fully differentiated perivascular DC are found in close association with T cells and with B cell follicles, sometimes containing follicular DC. These perivascular DC migrate across the activated endothelium from blood and receive differentiative signals within the joint from monocyte-derived cytokines and CD40-ligand+ T cells. In the SF, DC manifest an intermediate phenotype, similar to that of monocyte-derived DC in vitro. Like a delayed-type hypersensitivity response, the rheumatoid synovium represents an effector site. DC at many effector sites have a characteristic pattern of infiltration and differentiation. It is important to note that the effector response is not self-limiting in RA autoimmune inflammation. In this article, we argue that the presentation of self-antigen by DC and by autoantibody-producing B cells is critical for the perpetuation of the autoimmune response. Permanently arresting this ongoing immune response with either pharmaceutical agents or immunotherapy is a major challenge for immunology.

Functional CD40 ligand is expressed by T cells in rheumatoid arthritis.

CD40 ligand (CD40-L), a member of the tumor necrosis family of transmembrane glycoproteins, is rapidly and transiently expressed on the surface of recently activated CD4+ T cells. Interactions between CD40-L and CD40 induce B cell immunoglobulin production as well as monocyte activation and dendritic cell differentiation. Since these features characterize rheumatoid arthritis (RA), the expression and function of CD40-L in RA was examined. Freshly isolated RA peripheral blood (PB) and synovial fluid (SF) T cells expressed CD40-L mRNA as well as low level cell surface CD40-L. An additional subset of CD4+ RA SF T cells upregulated cell surface CD40-L expression within 15 min of in vitro activation even in the presence of cycloheximide, but soluble CD40-L was not found in SF. CD40-L expressed by RA T cells was functional, since RA PB and SF T cells but not normal PB T cells stimulated CD40-L-dependent B cell immunoglobulin production and dendritic cell IL-12 expression in the absence of prolonged in vitro T cell activation. In view of the diverse proinflammatory effects of CD40-L, this molecule is likely to play a central role in the perpetuation of rheumatoid synovitis. Of importance, blockade of CD40-L may prove highly effective as a disease modifying therapy for RA.

Nuclear localization of RelB is associated with effective antigen-presenting cell function.

Dendritic cells (DC) are potent APCs that enter resting tissues as precursors and, after Ag exposure, differentiate and migrate to draining lymph nodes. The phenotype of RelB knockout mice implicates this member of the NF kappa B/Rel family in DC differentiation. To further elucidate the role of RelB in DC differentiation, mRNA, intracellular protein expression, and DNA binding activity of RelB were examined in immature and differentiated human DC, as well as other PB mononuclear cell populations. RelB protein and mRNA were detected constitutively in lymphocytes and in activated monocytes, differentiated DC, and monocyte-derived DC. Immunohistochemical staining demonstrated RelB within the differentiated lymph node interdigitating DC and follicular DC, but not undifferentiated DC in normal skin. Active nuclear RelB was detected by supershift assay only in differentiated DC derived from either PB precursors or monocytes and in activated B cells. These RelB+ APC were potent stimulators of the MLR. The data indicate that RelB expression is regulated both transcriptionally and post-translationally in myeloid cells. Within the nucleus, RelB may specifically transactivate genes that are critical for APC function.

Olfactory neuronal cell lines generated by retroviral insertion of the n-myc oncogene display different developmental phenotypes.

Being genetically homogeneous, clonal cell lines are potentially important for investigating many aspects of cellular differentiation. We describe here the creation of clonal cell lines by immortalization of neuronal precursor cells from the adult mouse olfactory epithelium. Unlike neurons elsewhere in the vertebrate nervous system, the olfactory sensory neuron can be replaced throughout the lifespan of the animal. However, little is known about the molecular aspects of olfactory neurogenesis. Continuous cell lines were generated by retroviral transduction of the n-myc proto-oncogene into the mitotically active basal cells of the olfactory epithelium which give rise to the sensory neuron. Twenty-one clonal cell lines were produced which could be divided into three distinct morphological classes: one with flat, epithelial-like cells only; another with round, flat, and bipolar cells; and a third with large flat and large bipolar cells. These morphological classes had different patterns of intermediate filament expression, as shown by immunocytochemistry and immunoblot analysis. All cells in all cell lines expressed the intermediate filament protein vimentin. Most bipolar cells, but not other cell types, expressed neurofilament protein and in one morphological class the bipolar cells co-expressed neurofilament and glial fibrillary acidic protein. Several cell lines expressed mRNA for OMP, a marker of mature olfactory sensory neurons, and GOLF, a guanine nucleotide binding protein involved in olfactory sensory transduction. It is concluded that these cell lines were immortalized from sensory neuron precursors late in the lineage pathway. Other cell lines appear to have been immortalized at earlier stages in the lineage pathway. These cell lines therefore provide useful tools for the investigation of neuronal differentiation and sensory transduction in the olfactory epithelium.

Neurogenesis in adult human.

This report describes neurogenesis in the adult human olfactory epithelium in vitro. Olfactory epithelium was collected at autopsy and by biopsy, and grown in serum-free medium. Basic fibroblast growth factor induced the differentiation of bipolar cells which were immunopositive for several neuronal proteins but not glial proteins. [3H]thymidine autoradiography confirmed that these neurones were born in vitro. The results demonstrate that the adult human olfactory epithelium retains the capacity for neurogenesis and neuronal differentiation, at least until the age of 72 years. It is now possible to examine neurones and neurogenesis in biopsies from patients with disorders that may involve a neurodevelopmental or neurodegenerative aetiology such as schizophrenia, bipolar disorder and Alzheimer's disease.

FGF2 promotes neuronal differentiation in explant cultures of adult and embryonic mouse olfactory epithelium.

Neurogenesis in the adult olfactory epithelium is highly regulated in vivo. Little is known of the molecular signals which control this process, although contact with the olfactory bulb or with astrocytes has been implicated. Explants of mouse olfactory epithelium were grown in the presence or absence of several peptide growth factors. Basic fibroblast growth factor (FGF2) stimulated differentiation of sensory neurons in adult and embryonic olfactory epithelium. Other growth factors tested were ineffective. FGF2-stimulated neurons were born in vitro and expressed neurofilament, neural cell adhesion molecule, and beta-tubulin. The cells also expressed olfactory marker protein, a marker for mature olfactory sensory neurons in vivo. These bipolar neurons did not express glial fibrillary acidic protein or low-affinity nerve growth factor receptor. These results indicate that neither astrocytes nor olfactory bulb are necessary for differentiation of olfactory sensory neurons in vitro.