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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.
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 14 -15 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).
RESULTS
A.
DCGM-CSF
DCIL-10
25
11
23
17
CD80
21
22
CD86
21
24
MHC-Il
CYTOKINE SECRETION
21
600
41
62
25
37
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 DCIL-10,
DCGM-CSF or DCTNF on day 42 (Schneider et al, 2001).
BALB/c mice with severe ALD (≈60% airway
25
eosinophils on airway allergen challenge)
6
were given 1x10 DCIL-10, DCGM-CSF, or DCTNF
0
transtracheally. Over the next 3 weeks they -25
were assess by head-out body plethysmo-50
graphy for AHR to methacholine. This
experiment is representative of ≈8 others in -75
which we have found that, beginning at 15 -17-100
0mg
days post-transplant, the AHR of DCIL-10treated mice, but not those treated with either
DCGM-CSF or DCTNF, disappears.
28
0
41
FACS analysis also confirmed that the DCIL-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
IL-6
IL-10
IL-12
TGF-b
900
DCIL-10
(at 40C)
DCIL-10
DCGMCSF
DCTNF
ALD
-25
ALD+Saline
-50
Ald+DC GM -CSF
-50
DCIL-10
ALD+Saline
DCIL-10
-100
0mg 1.5mg 6mg 25mg
-100
Ald+DC GM -CSF
ALD+DC IL-10
ALD+DC IL-10
ALD+DC TNF
-75
ALD+DC TNF
0mg 1.5mg 6mg 25mg
Normal
ALD
ALD+Saline
ALD+DC GM-CSF
ALD+DC IL-10
ALD+DC TNF
sal
DC
DC/OVA
med
IL-12
IL-10
80
200
40
100
0
sal
IL-9
DC
DC/OVA
0
med
200
100
sal
DC
DC/OVA
0
0
med
sal
TGF-b
IL-13
DC
DC/OVA
med
IFN-g
120
120
200
300
80
200
0
sal
DC
DC/OVA
med
40
40
100
600
Norm.
300
80
sal
DC
DC/OVA
med
0
sal
DC
DC/OVA
med
0
sal
DC
DC/OVA
med
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: 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.
-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 212:101-109
300
0
0
Both DCIL-10 and DCGM-CSF avidly phagocytosed FITC-dextran, while
the DCTNF did not.
Normal
ALD
Methacholine (mg/ml)
120
200
100
Number of cells
(at 370C)
Normal
-25
-75
1.5mg 6mg 25mg
IL-5
IL-4
300
CHEMOKINE RECEPTORS
FITC-dex:
100 µg/ml
50 µg/ml
DCIL-10
DCIL-10
DAY 21
0
0
ALD
100
No FITC-dex
25
DAY 14
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
providing additional protective effects over DCIL-10 not exposed to OVA. (These results comprise one
representative experiment of eight)
DCGM-CSF and DCTNF populations did not express appreciable levels of IL-10
or TGFb. DCTNF expressed high levels of TNF and IL-12, while DCGM-CSF
expressed substantial amounts of IL-6, but not the other cytokines.
FITC-dextran PHAGOCYTOSIS
25
FIG. 3. DCIL-10 THERAPY REDUCES Th2-CYTOKINES IN THE AIRWAYS.
IL-1b
B.
DAY 7
400
200
DCTNF
ALLERGIC LUNG DISEASE (ALD) MODEL
Flow@50%TVe1
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 DCIL-10 were compared in the FACS analysis with immature DC GM-CSF and
immunostimulatory, mature DCTNF. DCIL-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).
CD54
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.
FIG 2. DCIL-10-TREATMENTS ABROGATE AHR IN MICE WITH SEVERE ALD.
FIG. 1. FUNCTIONAL CHARACTERIZATION OF DCIL-10 IN VITRO
CD40
HYPOTHESIS
TOLEROGENIC BONE MARROW-DERIVED DENDRITIC CELLS CAN BE
USED AS AN EFFECTIVE THERAPY FOR SEVERE ALLERGIC LUNG
DISEASE
OBJECTIVES
0.1
1
10
MIP-1a (ng/ml)
100
In MIP-3a chemotaxis (i.e., CCR7) assays, DCGM-CSF and DCIL-10 were shown to express
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
RESEARCH AND THE SASKATCHEWAN LUNG ASSOCIATION
CONCLUSION: DELIVERY OF DCIL-10, BUT NOT DCGMCSF OR DCTNF, INTO THE AIRWAYS OF MICE WITH ALD
ABROGATES AHR & SUBSTANTIALLY AMELIORATES
Th2 REACTIVITY