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					                                                        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.
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                                                                                                          TOLEROGENIC BONE MARROW-DERIVED DENDRITIC CELLS CAN BE
significant pharmacological advances in asthma therapy, the past two decades have seen an alarming increase                                                                                                        USED AS AN EFFECTIVE THERAPY FOR SEVERE ALLERGIC LUNG
in the prevalence of asthma world wide. In the United States alone, asthma affects approximately 14 -15 million
children and adults.                                                                                                                                                                                               DISEASE
    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
                                                                                                                                                                                                                      1. To generate (through culture in IL-10-containing media) and characterize
types of antigens are unmatched in this context. The decision of whether or not encounter with an antigen will                                                                                                         tolerogenic populations of mouse bone marrow-derived dendritic cells (DC)
lead to an immune response is controlled in many respects at the level of APC and is subject to tight regulation.                                                                                                     2. To assess the impact of these DCs, as well as fully mature BMDC, on
Tolerogenic DC have been implicated as critical in defining immunologic „self‟ and preventing the induction of
                                                                                                                                                                                                                       tolerance induction in a mouse model of allergic lung disease/asthma.
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                                                                                                                                                               ALLERGIC LUNG DISEASE (ALD) MODEL
expression of co-stimulatory molecules, and can lead to suppression of antigen specific responses. We wished                                                                                                           BALB/c mice were sensitized with OVA/alum (2 µg/mg, i.p.) on dy 0 & 14,
to determine whether such “tolerogenic” DC could reverse pre-existing asthma using a standard model of ALD                                                                                                             exposed to 1% OVA aerosols on days 28, 30, & 32, then treated with DCIL-10,
(Schneider et al, 2001).                                                                                                                                                                                               DCGM-CSF or DCTNF on day 42 (Schneider et al, 2001).

RESULTS                                                                                                                                                                                                              FIG 2. DCIL-10-TREATMENTS ABROGATE AHR IN MICE WITH SEVERE ALD.
FIG. 1. FUNCTIONAL CHARACTERIZATION OF DCIL-10 IN VITRO                                                                                                                                                       BALB/c mice with severe ALD (≈60% airway
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                                                                                                                            25
dy, the cells were analyzed (A) by FACS for multiple markers (left panel), in vitro cytokine release (right panel), (B) phagocytic capacity
                                                                                                                                                                                                              eosinophils on airway allergen challenge)                                                           DAY 7                             25                                           25
                                                                                                                                                                                                              were given 1x10  6 DC                                                                                                                                   DAY 14
                                                                                                                                                                                                                                   IL-10, DCGM-CSF, or DCTNF                                                                                                                                                         DAY 21

(left) and chemokine receptor expression (right). These DCIL-10 were compared in the FACS analysis with immature DC GM-CSF and                                                                                                                                  0
                                                                                                                                                                                                                                                                                                                                                     0                                               0
                                                                                                                                                                                                                                                                                                                                                                                                  Normal                                Normal

immunostimulatory, mature DCTNF. DCIL-10 expressed slightly lower levels of cell surface CD40, CD54 and MHC-II. They also released                                                                            transtracheally. Over the next 3 weeks they -25                                                                                                                                     ALD                                   ALD

significant amounts of IL-10 and TGFb (A). They possessed functional phagocytosis and strong chemotaxis to the inflammatory                                                                                                                                                                                                                         -25                                         -25
                                                                                                                                                                                                              were assess by head-out body plethysmo-                                                                                                                                             ALD+Saline                            ALD+Saline

chemokine MIP-1a (B).                                                                                                                                                                                         graphy for AHR to methacholine. This            -50                                                                                   -50                                         -50 DC GM-CSF
                                                                                                                                                                                                                                                                                                                                                                                                                              DCIL-10   Ald+ DC GM-C SF

                                                                                                                                                                                                                                                                                                                                                                                      DCIL-10     ALD+DC IL-10                          ALD+DC IL-10

                                                                                                                                                                                                              experiment is representative of ≈8 others in -75
                                                                                                                                                                                                                                                                                                                                                    -75                                         -75 TNF
                                                                                                                                                                                                                                                                                                                                                                                                  ALD+DC                                ALD+DC TNF

  A.                       CD40             CD54        CD80             CD86         MHC-Il                                        CYTOKINE SECRETION                                                        which we have found that, beginning at 15 -17  -100                                                                                  -100                                     -100
                                                                                                                                                                                                              days post-transplant, the AHR of DCIL-10-           0mg                                       1.5mg 6mg 25mg                                0mg 1.5mg 6mg 25mg                             0mg 1.5mg 6mg 25mg
       DCGM-CSF           25                11          21              21
                                                                                       21                               600                                                                                   treated mice, but not those treated with either                                                                       Methacholine (mg/ml)
                                                                                                                                                                                                              DCGM-CSF or DCTNF, disappears.                                                                           Normal                                      ALD                               ALD+Saline

                                                                                                                        400                                                                                                                                                                                            ALD+DC GM -CSF                              ALD+DC IL-10                      ALD+DC TNF
       DCIL-10             23               17           22             24             28
                                                                                                                                                                                                                     FIG. 3. DCIL-10 THERAPY REDUCES Th2-CYTOKINES IN THE AIRWAYS.
                                                                                                                                                                                                                    BAL fluids from ALD mice treated with saline, DCIL-10 , or OVA-pulsed DCIL-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
                           41                            25            37              41                                    0                                                                                      providing additional protective effects over DCIL-10 not exposed to OVA. (These results comprise one
       DCTNF                                 62
                                                                                                                                        IL-1b       IL-6        IL-10         IL-12       TGF-b                     representative experiment of eight)
                                                                                                                                                                                                                                                                                                                                                                                  ALD                                 Norm.
                   FACS analysis also confirmed that the DCIL-10 populations did not express neutrophil,                         DCGM-CSF and DCTNF populations did not express appreciable levels of IL-10                                    IL-4                                IL-5                                                        IL-10                                             IL-12
                   macrophage, B or T cell markers, and that they did express low levels of CD11c, and                           or TGFb. DCTNF expressed high levels of TNF and IL-12, while DCGM-CSF                      300                                                                                       300
                   DEC205, as well as expected levels of CD11b, MHC-I & CD45RB                                                   expressed substantial amounts of IL-6, but not the other cytokines.                                                                 120
                                                                                                                                                                                                                            200                                                                                       200
                                                                                                                                                                                                                            100                                                                                       100                                                 100

  B.                                                                                                                                                                                                                                    sal    DC     DC/OVA   med
                                                                                                                                                                                                                                                                           sal    DC              DC/OVA    med
                                                                                                                                                                                                                                                                                                                                  sal          DC         DC/OVA    med
                                                                                                                                                                                                                                                                                                                                                                                  sal           DC         DC/OVA    med
                   FITC-dextran PHAGOCYTOSIS                                                                                 CHEMOKINE RECEPTORS                                                                                               IL-9                               IL-13                                                        TGF-b                                              IFN-g
                                                                                                                                 DCGMCSF                DCIL-10               DCTNF                                   200
                              No FITC-dex                  FITC-dex:                                                   900                                                                                                                                                                                                                                                 80
                                                   100 µg/ml         50 µg/ml                                                                                                                                                                                        200
                                                                                                                                                                                                                                                                                                                       40                                                  40
                                                                                                     Number of cells

                                                                                                                                                                                                                        0                                              0                                                  0                                                 0
       (at 370C)                                                                                                       600                                                                                                        sal         DC    DC/OVA   med            sal   DC              DC/OVA   med                    sal         DC          DC/OVA   med          sal             DC         DC/OVA   med

                                                                                                                                                                                                                     -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: 394-400.
                                                                                                                                                                                                                     -Steinbrink K, Matthias W, Jonuleit H, Jurgen K and Enk AH (1997) Induction of tolerance by IL-10 treated dendritic cells. J Immunol 159: 4772-80.
        DCIL-10                                                                                                                                                                                                      -Schneider AM, Zhang X, Li F, and Gordon JR. (2001) Differential induction of allergen-specific IgA, AHR, or allergic airway disease following sensitization with
        (at 40C)                                                                                                                                                                                                     limiting doses of ovalbumin-alum. Cell Immunol 212:101-109

                       Both DCIL-10 and DCGM-CSF avidly phagocytosed FITC-dextran, while
                                                                                                                                    0       0.1         1      10
                                                                                                                                                  MIP-1a (ng/ml)
                                                                                                   In MIP-3a chemotaxis (i.e., CCR7) assays, DCGM-CSF and DCIL-10 were shown to express
                                                                                                                                                                                                                        CONCLUSION: DELIVERY OF DCIL-10, BUT NOT DCGM-
                                                                                                                                                                                                                        CSF OR DCTNF, INTO THE AIRWAYS OF MICE WITH ALD
                       the DCTNF did not.                                                          low, but significant, levels of CCR7, while the DCTNF responded very strongly via this
                                                                                                   receptor, as expected (data not shown).

                                                                                                                                                                                                                        ABROGATES AHR & SUBSTANTIALLY AMELIORATES
                                                                                                                                                                                                                        Th2 REACTIVITY

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