Annemarie van Nieuwenhuijze

GM-CSF increases cross-presentation and CD103 expression by mouse CD8⁺ spleen dendritic cells.

Resident CD8+ DCs perform several functions, including cross‐presenting antigen and rapidly engulfing the Gram‐positive intracellular pathogen Listeria monocytogenes. Little is known about how these functions of CD8+ DCs are modulated. Here, we show that granulocyte‐macrophage CSF (GM‐CSF), a cytokine that exists at low levels at steady state but is elevated during infection and inflammation, enhances cross‐presentation and rapid uptake of L. monocytogenes by resident CD8+ DCs. This previously unrecognized functional enhancement of CD8+ DCs by GM‐CSF was independent of promoting DC survival in vitro. Enhancement of these functions by GM‐CSF was also marked by CD103 expression on CD8+ DCs that was strongly regulated by GM‐CSF. Our findings not only identify GM‐CSF as a key molecule regulating CD8+ DC function, but also as a factor responsible for functional heterogeneity of CD8+ DCs that is at least substantially demarcated by CD103 expression.

Differentiation of Inflammatory Dendritic Cells Is Mediated by NF-κB1–Dependent GM-CSF Production in CD4 T Cells

Rel/NF-κB transcription factors regulate inflammatory and immune responses. Despite possible subunit redundancy, NF-κB1–deficient (Nfkb1−/−) mice were profoundly protected from sterile CD4 T cell-dependent acute inflammatory arthritis and peritonitis. We evaluated CD4 T cell function in Nfkb1−/− mice and found increased apoptosis and selectively reduced GM-CSF production. Apoptosis was blocked by expression of a Bcl-2 transgene without restoring a disease response. In contrast with wild-type cells, transfer of Nfkb1−/− or GM-CSF–deficient CD4 T cells into RAG-1–deficient (Rag1−/−) mice failed to support arthritis induction. Injection of GM-CSF into Nfkb1−/− mice fully restored the disease response, suggesting that T cells are an important source of GM-CSF during acute inflammation. In Ag-induced peritonitis, NF-κB1–dependent GM-CSF production in CD4 T cells was required for disease and for generation of inflammatory monocyte-derived dendritic cells (MoDC), but not conventional dendritic cells. MoDC were identified in inflamed synovium and draining lymph nodes during arthritis. These MoDC produced high levels of MCP-1, a potent chemoattractant for monocytes. This study revealed two important findings: NF-κB1 serves a critical role in the production of GM-CSF by activated CD4 T cells during inflammatory responses, and GM-CSF derived from these cells drives the generation of MoDC during inflammatory disease.

GM-CSF as a therapeutic target in inflammatory diseases.

GM-CSF is a well-known haemopoietic growth factor that is used in the clinic to correct neutropaenia, usually as a result of chemotherapy. GM-CSF also has many pro-inflammatory functions and recent data implicates GM-CSF as a key factor in Th17 driven autoimmune inflammatory conditions. In this review we summarize the findings that have led to the development of GM-CSF antagonists for the treatment of autoimmune diseases like rheumatoid arthritis (RA) and discuss some results of recent clinical trials of these agents.

Promotion of the local differentiation of murine Th17 cells by synovial macrophages during acute inflammatory arthritis.

OBJECTIVE To examine the generation of proinflammatory Th17 cells at the site of tissue inflammation and in draining lymph nodes using an interleukin-17 (IL-17)-dependent model of acute inflammatory arthritis. METHODS Arthritis was elicited in mice by intraarticular injection of methylated bovine serum albumin (mBSA) into the knee and subcutaneous injection of IL-1beta. Anti-IL-17 or control antibodies were administered during arthritis induction. Cytokine expression was evaluated by intracellular cytokine staining of synovial lymphocytes, by polymerase chain reaction analysis of RNA extracted from lymph node cells, and by enzyme-linked immunosorbent assay of cell culture supernatants. Th17 differentiation of naive CD4+ T cells was assessed in cocultures with macrophages from arthritic mice. RESULTS Anti-IL-17 antibody administered during acute arthritis markedly reduced disease, indicating that the model is IL-17 dependent. IL-17 messenger RNA (mRNA), but not protein, was detected in draining lymph node CD4+ T cells and preceded joint inflammation. In addition, mRNA for Th17 cell-stimulatory cytokines (transforming growth factor beta, IL-6) and Th17 cell-inhibitory cytokines (interferon-gamma, IL-4) was detected in lymph nodes following injection of mBSA and IL-1beta. Th17 cells were clearly identified in the inflamed synovium at the peak of disease. Synovial macrophages supported Th17 cell generation from naive CD4+ T cell precursors stimulated via CD3 in vitro and produced high levels of IL-6. In contrast, peritoneal macrophages failed to induce Th17 cell differentiation and produced less IL-6. CONCLUSION These results suggest that Th17 cell differentiation is initiated in draining lymph nodes but that IL-17-producing cells are restricted to the inflamed synovium, being generated in response to local cytokines produced by inflammatory macrophages.

Autoimmune regulator controls T cell help for pathogenetic autoantibody production in collagen‐induced arthritis

OBJECTIVE Autoimmune regulator (Aire) promotes the ectopic expression of tissue-restricted antigens in medullary thymic epithelial cells (mTECs), leading to negative selection of autoreactive T cells. This study was undertaken to determine whether loss of central tolerance renders Aire-deficient (Aire-/-) mice more susceptible to the induction of autoimmune arthritis. METHODS Medullary TECs were isolated from Aire-/- and wild-type C57BL/6 mice for gene expression analysis. Collagen-induced arthritis (CIA) was elicited by injection of chick type II collagen (CII) in adjuvant. Cellular and humoral immune responses to CII were evaluated. Chimeric mice were created by reconstituting lymphocyte-deficient mice with either Aire-/- or wild-type CD4 T cells and wild-type B cells. RESULTS Wild-type, but not Aire-/-, mTECs expressed the CII gene Col2a1. Aire-/- mice developed more rapid and severe CIA, showing elevated serum anti-CII IgG levels, with earlier switching to arthritogenic IgG subclasses. No evidence was found of enhanced T cell responsiveness to CII in Aire-/- mice; however, Aire-/- CD4 T cells were more efficient at stimulating wild-type B cells to produce anti-CII IgG following immunization of chimeric mice with CII. CONCLUSION Our findings indicate that Aire-dependent expression of CII occurs in mTECs, implying that there is central tolerance to self antigens found in articular cartilage. Reduced central tolerance to CII in Aire-/- mice manifests as increased CD4 T cell help to B cells for cross-reactive autoantibody production and enhanced CIA. Aire and central tolerance help prevent cross-reactive autoimmune responses to CII initiated by environmental stimuli and limit spontaneous autoimmunity.

Evaluation of the Bcl-2 family antagonist ABT-737 in collagen-induced arthritis

Therapeutic manipulation of cellular apoptosis holds great promise for malignant and potentially nonmalignant diseases. A relative resistance to apoptosis in RA synovium is associated with increased expression of prosurvival Bcl‐2 family members. In this study, we demonstrate that treatment of DBA/1 mice, prior to the onset of CIA with ABT‐737, a BH3 mimetic targeting Bcl‐2, Bcl‐w, and Bcl‐xL, ameliorated disease development. In contrast, treatment of mice with ABT‐737 in established CIA did not alter the course of disease. ABT‐737 induced lymphopenia, however pathogenic lymphoid populations in CIA mice were less affected, as shown by relatively normal T and B cell responses to CII. Naïve lymphocytes were highly sensitive to apoptosis after culture with ABT‐737, but synovial macrophages and neutrophils were not. Mcl‐1 was detected in synovial monocyte/macrophages and neutrophils and strikingly, its expression, rather than Bcl‐2 and Bcl‐xL, increased in the affected paws and lymphoid organs of mice with CIA. These observations implicate Mcl‐1, which is not targeted by ABT‐737, in the survival of inflammatory cells in established CIA and suggest that antagonism of Mcl‐1 may be more effective in diseases such as RA.

Bcl‐2 overexpression ameliorates immune complex‐mediated arthritis by altering FcγRIIb expression and monocyte homeostasis

RA is a chronic autoimmune disease characterized by accumulation of inflammatory cells within synovial joints. RA is associated with a failure of apoptosis of infiltrating leukocytes, thought to be a result of overexpression of prosurvival Bcl‐2 proteins. Overexpression of Bcl‐2 in hematopoietic cells can result in spontaneous autoimmunity. We therefore hypothesized that increased Bcl‐2 in the hematopoietic compartment would reduce apoptosis and thereby, exacerbate inflammatory arthritis. Paradoxically, we found that overexpression of Bcl‐2 in mice (vav‐bcl‐2) markedly reduced pathology in antibody‐dependent models of RA (CIA and K/BxN serum transfer arthritis). No such protection was observed in a model of CD4+ T cell‐dependent, B cell‐independent arthritis (mBSA/IL‐1‐induced arthritis). In CIA, vav‐bcl‐2 Tg mice had lower antibody production to CII, which might explain reduced disease. However, Bcl‐2 overexpression also reduced passive K/BxN serum transfer arthritis. Overexpression of Bcl‐2 caused a monocytosis, with preferential expansion of Ly6Clo monocytes and increased expression of the inhibitory receptor for IgG, FcγRIIb, on leukocytes. Skewing of the myeloid cell population, increases in FcγRIIb, and reduced arthritis were independent of the hypergammaglobulinemia found in vav‐bcl‐2 Tg mice. These data reveal selective effects of the Bcl‐2‐regulated apoptotic pathway on monocyte differentiation and the expression of FcRs critical for regulation of antibody/immune complex‐mediated disease.