TOLEROGENIC BONE MARROW-DERIVED DENDRITIC CELLS MODULATE ALLERGIC REACTIVITY OF LUNG CELLS FROM MICE WITH SEVERE ALLERGIC LUNG DISEASE. Aarti Nayyar, Xiaobei Zhang and John R. Gordon. Immunology Research Group (Dept Vet Microbiology), University of Saskatchewan, Saskatoon. TOLEROGENIC BONE MARROW-DERIVED DENDRITIC CELLS MODULATE ALLERGIC REACTIVITY OF LUNG CELLS FROM MICE WITH SEVERE ALLERGIC LUNG DISEASE. Aarti Nayyar, Xiaobei Zhang and John R. Gordon. Immunology Research Group (Dept Vet Microbiology), University of Saskatchewan, Saskatoon. BACKGROUND Asthma (allergic lung disease; ALD) is presently described as a syndrome characterized by: (a) intermittent and reversible airway obstruction; (b) airway hyperresponsiveness (AHR); and (c) airway inflammation. Despite significant pharmacological advances in asthma therapy, the past two decades have seen an alarming increase in the prevalence of asthma world wide. In the United States alone, asthma affects approximately million children and adults. Dendritic cells (DC) are a family of professional antigen (Ag) presenting cells (APC), considered by many to be the central APC for induction of primary immune responses. Their abilities to process and present various types of antigens are unmatched in this context. The decision of whether or not encounter with an antigen will lead to an immune response is controlled in many respects at the level of APC and is subject to tight regulation. Tolerogenic DC have been implicated as critical in defining immunologic ‘self’ and preventing the induction of both autoimmunity and chronic inflammation against environmental proteins. Recent studies show that different subsets of DC play important roles in central and peripheral tolerance. It has been reported that treatment of DC with IL-10 inhibits their terminal differentiation, can reduce expression of co-stimulatory molecules, and can lead to suppression of antigen specific responses. We wished to determine whether such “tolerogenic” DC could reverse pre-existing asthma using a standard model of ALD (Schneider et al, 2001). Asthma (allergic lung disease; ALD) is presently described as a syndrome characterized by: (a) intermittent and reversible airway obstruction; (b) airway hyperresponsiveness (AHR); and (c) airway inflammation. Despite significant pharmacological advances in asthma therapy, the past two decades have seen an alarming increase in the prevalence of asthma world wide. In the United States alone, asthma affects approximately million children and adults. Dendritic cells (DC) are a family of professional antigen (Ag) presenting cells (APC), considered by many to be the central APC for induction of primary immune responses. Their abilities to process and present various types of antigens are unmatched in this context. The decision of whether or not encounter with an antigen will lead to an immune response is controlled in many respects at the level of APC and is subject to tight regulation. Tolerogenic DC have been implicated as critical in defining immunologic ‘self’ and preventing the induction of both autoimmunity and chronic inflammation against environmental proteins. Recent studies show that different subsets of DC play important roles in central and peripheral tolerance. It has been reported that treatment of DC with IL-10 inhibits their terminal differentiation, can reduce expression of co-stimulatory molecules, and can lead to suppression of antigen specific responses. We wished to determine whether such “tolerogenic” DC could reverse pre-existing asthma using a standard model of ALD (Schneider et al, 2001). TOLEROGENIC BONE MARROW-DERIVED DENDRITIC CELLS CAN BE USED AS AN EFFECTIVE THERAPY FOR SEVERE ALLERGIC LUNG DISEASE 1. To generate (through culture in IL-10-containing media) and characterize tolerogenic populations of mouse bone marrow-derived dendritic cells (DC) 2. To assess the impact of these DCs, as well as fully mature BMDC, on tolerance induction in a mouse model of allergic lung disease/asthma. BALB/c mice were sensitized with OVA/alum (2 µg/mg, i.p.) on dy 0 & 14, exposed to 1% OVA aerosols on days 28, 30, & 32, then treated with DC IL-10, DC GM-CSF or DC TNF on day 42 (Schneider et al, 2001). HYPOTHESIS OBJECTIVES FIG 2. DC IL-10 -TREATMENTS ABROGATE AHR IN MICE WITH SEVERE ALD. BALB/c mice with severe ALD (≈60% airway eosinophils on airway allergen challenge) were given 1x10 6 DC IL-10, DC GM-CSF, or DC TNF transtracheally. Over the next 3 weeks they were assess by head-out body plethysmo- graphy for AHR to methacholine. This experiment is representative of ≈8 others in which we have found that, beginning at days post-transplant, the AHR of DC IL-10 - treated mice, but not those treated with either DC GM-CSF or DC TNF, disappears. DAY 21 FIG. 3. DC IL-10 THERAPY REDUCES Th2-CYTOKINES IN THE AIRWAYS. BAL fluids from ALD mice treated with saline, DC IL-10, or OVA-pulsed DC IL-10 were assessed on treatment day 28. The levels of IL-4, IL-5, and IL-13 were significantly affected by the DC treatments, with OVA-presenting DC providing additional protective effects over DC IL-10 not exposed to OVA. (These results comprise one representative experiment of eight) ALD Norm. IL sal DC DC/OVA med IL sal DC DC/OVA med IL sal DC DC/OVA med IFN-  sal DC DC/OVA med IL sal DC DC/OVA med IL sal DC DC/OVA med IL sal DC DC/OVA med TGF-  sal DC DC/OVA med B. DC IL-10 (at 37 0 C) DC IL-10 (at 4 0 C) No FITC-dex FITC-dex: 100 µg/ml 50 µg/ml CONCLUSION: DELIVERY OF DC IL-10, BUT NOT DC GM- CSF OR DC TNF, INTO THE AIRWAYS OF MICE WITH ALD ABROGATES AHR & SUBSTANTIALLY AMELIORATES Th2 REACTIVITY Bone marrow cells from BALB/c mice were cultured in high (20 ng/ml), then low (7.5 ng/ml) dose GM-CSF + IL-10 (50 ng/ml). After 15 dy, the cells were analyzed (A) by FACS for multiple markers (left panel), in vitro cytokine release (right panel), (B) phagocytic capacity (left) and chemokine receptor expression (right). These DC IL-10 were compared in the FACS analysis with immature DC GM-CSF and immunostimulatory, mature DC TNF. DC IL-10 expressed slightly lower levels of cell surface CD40, CD54 and MHC-II. They also released significant amounts of IL-10 and TGFb (A). They possessed functional phagocytosis and strong chemotaxis to the inflammatory chemokine MIP-1a (B). FIG. 1. FUNCTIONAL CHARACTERIZATION OF DC IL-10 IN VITRO SUPPORTED BY GRANTS FROM THE CANADIAN INSTITUTES FOR HEALTH RESEARCH AND THE SASKATCHEWAN LUNG ASSOCIATION RESULTS CD40 DC GM-CSF DC IL-10 DC TNF CD54 CD80 CD86 MHC-Il CYTOKINE SECRETION TGF-  DC GMCSF DC IL-10 DC TNF MIP-1  (ng/ml) Number of cells REFERENCES -Jonuleit H, Schmitt KE, Steinbrink K and Enk AH(2001) Dendritic cells as a tool to induce anergic and regulatory T cells. Trends Immunol 22: Steinbrink K, Matthias W, Jonuleit H, Jurgen K and Enk AH (1997) Induction of tolerance by IL-10 treated dendritic cells. J Immunol 159: Schneider AM, Zhang X, Li F, and Gordon JR. (2001) Differential induction of allergen-specific IgA, AHR, or allergic airway disease following sensitization with limiting doses of ovalbumin-alum. Cell Immunol 212: ALLERGIC LUNG DISEASE (ALD) MODEL A. FACS analysis also confirmed that the DC IL-10 populations did not express neutrophil, macrophage, B or T cell markers, and that they did express low levels of CD11c, and DEC205, as well as expected levels of CD11b, MHC-I & CD45RB FITC-dextran PHAGOCYTOSIS CHEMOKINE RECEPTORS DC GM-CSF and DC TNF populations did not express appreciable levels of IL-10 or TGF . DC TNF expressed high levels of TNF and IL-12, while DC GM-CSF expressed substantial amounts of IL-6, but not the other cytokines. Both DC IL-10 and DC GM-CSF avidly phagocytosed FITC-dextran, while the DC TNF did not. In MIP-3  chemotaxis (i.e., CCR7) assays, DC GM-CSF and DC IL-10 were shown to express low, but significant, levels of CCR7, while the DC TNF responded very strongly via this receptor, as expected (data not shown). DC IL-10