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
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
IL-1b IL-6 IL-10 IL-12 TGF-b representative experiment of eight)
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
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
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
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).
SUPPORTED BY GRANTS FROM THE CANADIAN INSTITUTES FOR HEALTH
ABROGATES AHR & SUBSTANTIALLY AMELIORATES
RESEARCH AND THE SASKATCHEWAN LUNG ASSOCIATION