[Show abstract][Hide abstract] ABSTRACT: Antibodies to the pan-leukocyte adhesion-GPCR CD97 efficiently block neutrophil recruitment in mice, thereby reducing antibacterial host defense, inflammatory disease, and hematopoietic stem cell mobilization. Here, we investigated the working mechanism of the CD97 antibody 1B2. Applying sterile models of inflammation, intravital microscopy, and mice deficient for the CD97L CD55, the complement component C3, or the FcR common γ-chain, we show that 1B2 acts in vivo independent of ligand-binding interference by depleting PMN granulocytes in bone marrow and blood. Granulocyte depletion with 1B2 involved FcR but not complement activation and was associated with increased serum levels of TNF and other proinflammatory cytokines. Notably, depletion of granulocytes by CD97 antibody required acute inflammation, suggesting a mechanism of conditional, antibody-mediated granulocytopenia.
[Show abstract][Hide abstract] ABSTRACT: CD97 is a member of the EGF-TM7 family of adhesion G protein-coupled receptors (GPCRs) broadly expressed on leukocytes. CD97 interacts with several cellular ligands via its N-terminal epidermal growth factor (EGF)-like domains. To understand the biological function of CD97, monoclonal antibodies (mAbs) specific for individual EGF domains have been applied in a variety of in vivo models in mice, which represent different aspects of innate and adaptive immunity. Targeting CD97 by mAbs inhibited the accumulation of neutrophilic granulocytes at sites of inflammation thereby affecting antibacterial host defense, inflammatory disorders and stem cell mobilization from bone marrow. Interestingly, targeting CD97 did not impact antigen-specific (adaptive response) models such as delayed type hypersensitivity (DTH) or experimental autoimmune encephalomyelitis (EAE). However, collagen-induced arthritis (CIA), a model for rheumatoid arthritis, was significantly ameliorated suggesting therapeutic value of CD97 targeting. CD97-deficient mice are essentially normal at steady state except for a mild granulocytosis, which increases under inflammatory conditions. Comparison of the consequences of antibody treatment and gene targeting implies that CD97 mAbs actively inhibit the innate response presumably at the level of granulocyte or macrophage recruitment to sites of inflammation. Based on the collected data, we propose that the CD97 mAbs either activate CD97-mediated signal transduction via a yet unknown mechanism or act by inducing CD97 internalization, making CD97 unavailable for binding to its ligands and thereby blocking recruitment of neutrophils and possibly macrophages.
Advances in Experimental Medicine and Biology 01/2010; 706:128-37. DOI:10.1007/978-1-4419-7913-1_11 · 1.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: CD97 is a member of the EGF-TM7 family of adhesion class receptors, with a proposed role in inflammatory cell recruitment. Neutralization of murine CD97 with the anti-mCD97 mAb 1B2 was efficacious in prevention of murine collagen-induced arthritis, a model with features resembling rheumatoid arthritis. Here, the therapeutic potential of neutralizing CD97 in arthritis was studied with emphasis on the 1B2 pharmacokinetics. Mice with established arthritis were treated with anti-mCD97 or anti-TNF-alpha serum. Ab pharmacokinetics and biodistribution were studied in diseased and nondiseased mice using labeled 1B2. The impact of CD97 expression on Ab pharmacokinetics was studied using CD97 knockout mice. Treatment with 1B2 showed an efficacy comparable to anti-TNF-alpha treatment. Pharmacokinetic analysis of 1B2 in wild-type and CD97 knockout mice indicated a dose-dependent Ab clearance, due to specific interaction with CD97. Biodistribution studies showed accumulation of 1B2 in spleen and lung. In vitro studies using murine splenocytes revealed that CD97 when bound to Ab was internalized. Moreover, soluble CD97 was detected in the supernatant, suggesting Ag shedding. Finally, in arthritic mice, higher levels of soluble CD97 were found and 1B2 treatment led to specific targeting of inflamed paws, resulting in a higher clearance rate of 1B2 in arthritic mice than in wild-type mice. In conclusion, our data support a therapeutic value of CD97 neutralization in experimental arthritis. The pharmacokinetic profile of the 1B2 Ab illustrates the complexity of Ab elimination from an organism and stresses the importance of understanding Ag-Ab interactions when developing therapeutic mAbs.
The Journal of Immunology 10/2009; 183(6):4127-34. DOI:10.4049/jimmunol.0901253 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phosphatidylinositol 3-kinase-dependent activation of protein kinase B (PKB) has been observed in rheumatoid arthritis (RA) synovial tissue, and mechanisms that interfere with this process are protective in animal models of arthritis. PKB can regulate cell survival and proliferation via phosphorylation-dependent inactivation of forkhead box class O (FoxO) transcription factors. The present study was undertaken to examine whether FoxO transcription factors are differentially inactivated in RA synovial tissue, and whether this inactivation correlates with laboratory and clinical parameters of disease activity.
The expression and phosphorylation of FoxO family members were assessed in synovial biopsy tissue from 12 patients with RA and 9 patients with inflammatory osteoarthritis (OA), by immunohistochemistry and quantitative computer-assisted image analysis. Immunoblotting was used to assess the interleukin-1beta (IL-1beta)- and tumor necrosis factor alpha (TNFalpha)-induced phosphorylation of FoxO1 and FoxO4 in cultured fibroblast-like synoviocytes (FLS) and macrophages.
FoxO1, FoxO3a, and FoxO4 were expressed and phosphorylated in synovial tissue from both RA patients and OA patients. In RA synovial tissue, phosphorylation of FoxO1 was observed in both FLS and macrophages, FoxO3a in T lymphocytes, and FoxO4 in macrophages alone. Following stimulation with IL-1beta and TNFalpha, FoxO1 and FoxO4 were phosphorylated in both RA and OA FLS and synovial macrophages, respectively. Inactivation of FoxO4 was significantly enhanced in the RA as compared with the OA synovial sublining. There was a strong negative correlation between inactivation of FoxO4 in RA synovial tissue and increased serum C-reactive protein levels and a raised erythrocyte sedimentation rate in RA patients.
All 3 FoxO family members examined were phosphorylated in both RA and OA synovial tissue; in particular, inactivation of FoxO4 was significantly enhanced in macrophages from RA synovial tissue. Thus, cell-specific inactivation of FoxO family members appears to differentially regulate cell survival and proliferation in the RA synovium.