Dendritic cells (DCs) play an important role in induction of cellular immune responses. It
seems that DCs that reside in different organs may be distinct in their ability to induce
immune responses. This study was done to address the differences between spleen and liver
DCs in induction of immune response and/or tolerance.
CD11c+ DCs were separated from the liver and spleen of C57BL/6 mice and pulsed with
myelin oligodendrocyte glycoprotein (MOG) peptide 35-55. 6×105 MOG35-55 pulsed spleen
or liver DCs were injected in foot pad of different groups of mice. Control groups received
unpulsed DCs. After 5 days, the mononuclear cells (MNCs) of the regional lymph nodes
were isolated from immunized mice for cytokine assays and lymphocyte transformation test.
To study the immunologic or tolerogenic effects of DCs, three weeks after immunization of
mice with MOG pulsed liver or spleen DCs, experimental autoimmune encephalomyelitis
(EAE) was induced in DC-immunized mice by injection of MOG along with complete
Freund’s adjuvant. Our results showed that spleen DCs were more potent in stimulating
lymph node T cells as illustrated in lymphocyte transformation test. Moreover IL-10
production was higher in mice immunized with liver DCs compared with those immunized
with splenic DCs (p=0.017). However, no significant difference in IFN-8 production was
observed between two groups. We also found that liver DCs+MOG immunized mice
displayed a significantly delayed disease onset compared with spleen DCs+MOG immunized
mice and the control groups. The disease score was also milder in liver DCs immunized mice
compared with other groups.
It seems that the higher IL-10 production induced by the liver DCs may be one of the
main factors in down regulation of immune responses in this organ. It can be concluded also
that the liver DCs may inhibit the progress of EAE by shifting the cytokines profile.
Key Words: Cytokines Profile; Experimental Autoimmune Encephalomyelitis; Liver Dendritic Cells; Splenic Dendritic Cellsimmunized mice however this
difference was not statistically different While the
amount of IL-10 production by lymphocytes of liver
DCs immunized group was significantly higher than
spleen DCs immunized group (p=0.017) (Figure 2).
The ratio of IFN- to IL-10 in mice immunized with
MOG pulsed splenic DCs (15.6±2) and those
immunized with MOG pulsed liver DCs showed also a
significant difference (3.7±1, p=0.014).
Functional heterogeneity and functional plasticity of
DCs provide an effective strategy to manipulate the
immune response in a desirable way.24 There have been
some advances in the development of DC-based
immunostimulatory and immunosuppressive strategies,
which are potentially appropriate to the treatment of
cancer, autoimmune disease, allograft rejection, allergy
and graft versus host disease.25-28
DCs may promote T cell immunity or tolerance,
depending not only on their precursors, but also on the
microenvironmental factors among which they
differentiate.29,30 There are evidences that donorderived
DCs may play a role in the immune privilege of
Figure 2. Cytokine secretion by lymph node cells of mice
pre-immunized by MOG35-55 pulsed liver or spleen DCs.
female normal C57BL/6 mice were immunized with
spleen ( ) and liver () MOG35-55-pulsed DCs and
unpulsed DCs. Five days post-immunization, IFN- and
IL-10 secretion by MNCs isolated from the popliteal
lymph node of mice in the presence (+Ag) and absence (-
Ag) of MOG 35-55 peptide were detected by ELISA.
It is proposed that the function of liver DCs in
induction of the immune response is different from that
of their splenic counterparts.
Herein, we have analyzed the T cell stimulatory
capacity and influence on Th1 and Th2 cytokine
production of liver DCs compared with splenic DCs.
Our results showed that the ratio of IFN- to IL-10
secretions, as representatives of Th1 and Th2
cytokines, by MNCs from mice immunized with
splenic DCs was significantly higher than the same
ratio in mice immunized with liver DCs. It was
reported that DCs isolated from Peyer’s patches also
secrete higher levels of IL-10 than splenic DC and are
able to induce the differentiation of Th2 cells.16 This
findings suggest that high IL-10 production by liver
DCs may also contribute to induction of Th2 responses
and might be the mechanism underlying the capacity of
liver allografts to subvert host immune responses and
tolerance induction. It has been reported that resident
G. Mosayebi and SM. Moazzeni
168/ IRANIAN JOURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY Vol. 10, No. 3, September 2011
DCs are the main population of non-parenchymal liver
cells which are responsible for the tolerogenic
properties of the liver. These cells mediate
immunosuppression by production of antiinflammatory
cytokines such as IL-10 and TGF beta as
well as by expression of the negative co-stimulator for
T cell activation programmed cell death ligand-1 (PDL1).
There is a great deal of evidence that DCs in the
target organ are central to the immunopathogenesis of
EAE and other Th1-mediated autoimmune diseases.33
Therefore EAE was used as a model in this study to
address the role of spleen and liver DCs in induction of
immunity or tolerance. In our experimental system,
none of the spleen or liver DCs pulsed with the
MOG35-55 peptide could induce EAE in naïve
C57BL/6 mice by 3 weeks post-immunization. In our
previous study we showed that only after 3
subcutaneous injections of MOG pulsed bone marrow
derived DCs along with pertussis toxin, mice showed
mild clinical symptoms of EAE.34 However, Weir et
al. showed that intravenous injection of bone-marrow
derived dendritic cells presenting MOG35-55 peptide
into naïve C57BL/6 mice could induce EAE.35 These
finding indicates that DCs presenting a self-peptide
possess the ability to activate naïve self-reactive T cells
and induce autoimmune disease.
In contrast, Huang et liver of mice, as previously described by Woo
et al, 21 with the following modifications. Mice were
anesthetized with ketamine, swabbed with 70% ethanol
and an abdominal mid-line incision was performed.
The liver was perfused for 3 min in situ via the inferior
vena cava, using 20-30 ml ice-cold phosphate buffer
saline (PBS) and a 22G intravenous catheter.
Two ml of collagenase D solution (1mg/ml) in
RPMI-1640 was then injected. The liver was excised
immediately, diced into small pieces and digested in
collagenase solution (5 ml/liver) for 30 min at 37°C,
with constant stirring. The digested tissue was then
filtered through a 0.1 mm sterile wire mesh. Cells from
three to four livers were pooled.
Functional Differences of Spleen and Liver Dendritic Cells
Vol. 10, No. 3, September 2011 IRANIAN JOURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY /165
The cell suspension was then washed twice with
PBS or RPMI-1640 medium by centrifugation at 400g
for 5 min at 4°C. The final pellet was re-suspended in
10-15 ml RPMI medium and overlaid on a nycodenz
(Pharma AS, Oslo, Norway) gradient 14.5% (w/v) and
centrifuged at 600 g for 15 min at 4°C.
The recovered low density NPCs from the interface
were collected using a Pasteur pipette and was washed
as described above. To enrich the DCs, NPCs were
suspended in complete tissue culture medium (RPMI-
1640 containing 10% FCS) and incubated overnight
(18 hr) at 37°C, 5% CO2 in tissue culture petri dishes.
At the end of the incubation time non-adherent cells
were recovered and layered on a 3 ml column of 13%
(w/v) nycodenz and centrifuged at 600g for 15 min at
Low-density cells referred as “DC-enriched cells”,
were carefully separated from the interface, washed and
used for Ag pulsing and immunization of mice. Flow
cytometric analysis of these cells by anti-CD11c
antibody revealed their purity to be more than 70%.
In vitro Pulsing of DCs
Dendritic-enriched cells were incubated with MOG35-
55 peptide (MEVGWYRSPFSRVVHLYRNGK;
Diapharm Ltd, Russia) (200 μg/ml) for 4–6 h at 37°C and
then washed twice to remove the excess antigen.
Immunization of Mice with Peptide Pulsed DCs
6×105 spleen or liver MOG pulsed DCs in PBS
were injected into the hind footpads of naïve C57BL/6
mice (50 μl/foot pad). Control groups received
unpulsed spleen or liver DCs.
Five days post-immunization, mice in each group
were sacrificed and cell preparations were made from
their popliteal lymph nodes and used as a source of
primed lymph node cells in the cytokine assays and
lymphocyte transformation test.
To compare the immunity or tolerance induction
effects of the liver and spleen DCs other groups of
mice (n=6) were immunized s.c. with the same number
of spleen or liver MOG pulsed or unpulsed (controls)
DCs in the flank region. These mice were monitored
daily for EAE symptoms for 3 weeks and finally were
used for EAE induction by classical EAE induction
Cytokine Assay and Lymphocyte Transformation
Lymph node cells (2×105 cells/well) were cultured
in click medium (Sigma-Aldrich, USA) containing 1%
normal mouse serum and 200 μg/ml MOG35-55
peptide as antigen. All cultures were done in triplicates
with negative control wells containing no antigen. The
cells supernatants were harvested after 72 hours of
culture for analysis of IFN- and IL-10 concentrations
by a sandwich ELISA kit with sensitivity of 0.762
pg/ml and 4 pg/ml respectively (BD Biosciences,
For lymphocyte transformation test, the incubation
period was extended for 80 hours in CO2 incubator.
One μCi of [3H] thymidine (GE healthcare, Sweden)
was then added to each culture well and the incubation
period continued for 18 hours. The cultures were
harvested onto glass fiber filter paper and counted in a
liquid scintillation counter (Wallac 1410, GE
healthcare). Results were expressed as the mean counts
per minute (CPM) of the triplicate cultures.
Induction of EAE in DCs Immunized Mice
After three weeks follow up of pre-immunized mice
with MOG pulsed or unpulsed spleen and liver DCs,
when no clinical symptoms of EAE was observed, the
classical EAE was induced. These mice were
inoculated subcutaneous in the flank with 100μl of an
emulsion containing 200μg of the encephalitogenic
peptide MOG and equal volume of complete Freund’s
adjuvant (Sigma-Aldrich, USA) supplemented with 4
mg /ml mycobacterium tuberculosis H37RA (Difco,
USA). Mice were then injected intra-peritoneally with
400ng of pertussis toxin (Sigma-Aldrich, USA) on the
day of immunization; this was repeated 2 days later.
Clinical Evaluation of EAE
Mice were monitored daily and neurological
impairments were scored on an arbitrary clinical score
as follows: 0, no clinical sign; 1, partial loss of tail
tonicity; 2, complete loss of tail tonicity; 3, flaccid tail
and abnormal gait; 4, hind leg paralysis; 5, hind leg
paralysis with hind body paresis; 6, hind and foreleg
paralysis; 7, moribund or death. The day of onset of
disease was considered as mean clinical score of 1 for
each group and the relapse was defined when a mouse
developed an increase of the clinical score (more than
1) accompanied by weight loss.23
G. Mosayebi and SM. Moazzeni
166/ IRANIAN JOURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY Vol. 10, No. 3, September 2011
All results are presented here as Mean±SD of at
least six different experiments. U-Mann Whitney test
was used for evaluation of statistical differences
between the results. The changes in clinical scores in
each group were analyzed by Friedman test. P values
less than 0.05 were considered statistically significant.al. showed that pretreatment
of Lewis rats with a subcutaneous injection of
autoantigen-pulsed plastic adherent DCs induced
tolerance to EAE. However the same effect was not
observed when using non-adherent floating DCs.36 It
has been shown that targeted expression of MOG to
DCs can promote tolerance induction and manifest a
significant delay in the development of EAE.37
Therefore, to study the probable tolerogenic effect
of injected DCs, we induced classical EAE in liver and
spleen DC of pre-immunized mice. Our results showed
that there is a significant difference in the day of onset
of disease between animals immunized with MOG35-
55-pulsed liver DCs (17±1) compared with MOGpulsed
spleen DCs (9±1) and classical EAE control
group (11±1). The disease symptoms were also milder
in mice immunized with pulsed liver DCs compared
with mice immunized with pulsed spleen DCs and
DCs form a heterogeneous cell population.38 CD8+
DCs may play a role in maintaining peripheral
tolerance, whereas CD8- DCs appear to be important
for inducing immune responses.38,39 A difference in the
frequency and ratio of DC subtypes appears to be one
of the reasons for the difference in immune response in
various lymphoid tissues. However, a number of
reports have demonstrated that the ratio of CD8- DCs
to CD8+ DCs in the liver and spleen is almost
identical.40,41 We also did not observe a significant
difference in this ratio between liver and spleen (data
With reference to the similar percentage of
CD11c+/CD8+ DCs and CD11c+/CD8- DCs in the
spleen and the liver, it seems that microenvironmental
factors and their effect on DCs have a stronger effect
on the development of immune responses compared to
the phenotype of the DCs. In fact, it was shown that
liver sinusoidal endothelial cells impair the DCs ability
to induce the proliferation of naïve T cells in vitro via
an unknown mechanism.42
In agreement with the effects of microenvironment,
Xia et al. also used the liver fibroblastic stromal cells to
mimic the liver microenvironment and found that liver
stroma could induce the differentiation of DCs with
low CD11c, MHC II but high CD11b expression, high
IL-10, but low IL-12 secretion from Lin CD117 +
progenitors. Such regulatory DCs could inhibit T-cell
proliferation, induce apoptosis of the activated T cells,
and dampen the damage of autoimmune hepatitis.31
It has also been reported that the route of
immunization determines whether immunity or
tolerance is induced.43 Subcutaneous injection of DCs
pulsed with MBP was reported to induced tolerance to
EAE in rats, whereas intravenous injection of peptidepulsed
DCs in the mouse generated encephalolitogenic
T cells.35,44 We also injected pulsed spleen or liver DCs
via the intravenous route but this treatment could not
induce any signs of EAE (data not shown). However,
the inability of spleen or liver-pulsed DCs to induce
EAE may be due to the low number of DCs injected.
Although DCs are highly potent activators of T cell
immunity and a few DCs appear to be sufficient to
induce an immune response.
The frequency of auto-reactive T cells is another
important factor in emersion of autoimmune response.45
Our LTT results showed that spleen derived DCs are
more potent stimulator of T cells than liver DCs.
Indeed the intensity and day of onset of EAE
induction in mice pre-immunized by different DCs
were compatible with T cell stimulatory potential and
the induced cytokine profile by these cells. It is
Functional Differences of Spleen and Liver Dendritic Cells
Vol. 10, No. 3, September 2011 IRANIAN JOURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY /169
possible that splenic DCs increase EAE severity by
higher level of T cell stimulation and shifting the
response toward the Th1 type, while liver DCs down
regulate EAE intensity by shifting the response toward
the Th2 type.
It can be concluded that the ability of liver DCs to
induce immunity is different from that of splenic DCs
and this difference may contributes to immune
privilege of liver.
1. Steinman RM, Cohn ZA. Identification of a novel cell
type in peripheral lymphoid organs of mice. I.
Morphology, quantitation, tissue distribution. J Exp Med
2. Mellman I. Antigen processing and presentation by
dendritic cells: cell biological mechanisms. Adv Exp Med
Biol 2005; 560:63-7.
3. Granucci F, Zanoni I, Ricciardi-Castagnoli P. Central role
of dendritic cells in the regulation and deregulation of
immune responses. Cell Mol Life Sci 2008; 65(11):1683-
4. Gad M, Claesson MH, Pedersen AE. Dendritic cells in
peripheral tolerance and immunity. APMIS 2003; 111(7-
5. Carreno LJ, Riedel CA, Kalergis AM. Induction of
tolerogenic dendritic cells by NF-kappaB blockade and
Fcgamma receptor modulation. Methods Mol Biol 2011;
6. Matta BM, Castellaneta A, Thomson AW. Tolerogenic
plasmacytoid DC. Eur J Immunol 2010; 40(10):2667-76.
7. Vremec D, Pooley J, Hochrein H, Wu L, Shortman K.
CD4 and CD8 expression by dendritic cell subtypes in
mouse thymus and spleen. J Immunol 2000; 164(6):2978-
8. Dunne PJ, Moran B, Cummins RC, Mills KH.
CD11c+CD8alpha+ dendritic cells promote protective
immunity to respiratory infection with Bordetella
pertussis. J Immunol 2009; 183(1):400-10.
9. Reid SD, Penna G, Adorini L. The control of T cell
responses by dendritic cell subsets. Curr Opin Immunol
10. Coquerelle C, Moser M. DC subsets in positive and
negative regulation of immunity. Immunol Rev 2010;
11. Vremec D, Shortman K. Dendritic cell subtypes in mouse
lymphoid organs: cross-correlation of surface markers,
changes with incubation, and differences among thymus,
spleen, and lymph nodes. J Immunol 1997; 159(2):565-
12. Thomson AW, O'Connell PJ, Steptoe RJ, Lu L.
Immunobiology of liver dendritic cells. Immunology and
cell biology 2002; 80(1):65-73.
13. Lau AH, Thomson AW. Dendritic cells and immune
regulation in the liver. Gut 2003; 52(2):307-14.
14. Starzl TE. The "privileged" liver and hepatic
tolerogenicity. Liver Transpl 2001; 7(10):918-20.
15. Iwasaki A, Kelsall BL. Freshly isolated Peyer's patch, but
not spleen, dendritic cells produce interleukin 10 and
induce the differentiation of T helper type 2 cells. J Exp
Med 1999; 190(2):229-39.
16. Khanna A, Morelli AE, Zhong C, Takayama T, Lu L,
Thomson AW. Effects of liver-derived dendritic cell
progenitors on Th1- and Th2-like cytokine responses in
vitro and in vivo. J Immunol 2000; 164(3):1346-54.
17. Sumpter TL, Lunz JG, Demetris AJ, Thomson AW.
Molecular regulation of hepatic dendritic cell function
and its relation to liver transplant outcome.
Transplantation 2009; 88(3 Suppl):S40-4.
18. Pillarisetty VG, Miller G, Shah AB, DeMatteo RP. GMCSF
expands dendritic cells and their progenitors in
mouse liver. Hepatology 2003; 37(3):641-52.
19. Lu L, Woo J, Rao AS, Li Y, Watkins SC, Qian S, et al.
Propagation of dendritic cell progenitors from normal
mouse liver using granulocyte/macrophage colonystimulating
factor and their maturational development in
the presence of type-1 collagen. J Exp Med 1994;
20. Zarnani AH, Moazzeni SM, Shokri F, Salehnia M,
Dokouhaki P, Shojaeian J, et al. The efficient isolation of
murine splenic dendritic cells and their cytochemical
features. Histochem Cell Biol 2006; 126(2):275-82.
21. Woo J, Lu L, Rao AS, Li Y, Subbotin V, Starzl TE, et al.
Isolation, phenotype, and allostimulatory activity of
mouse liver dendritic cells. Transplantation 1994;
22. Mosayebi G, Ghazavi A, Salehi H, Payani MA, Khazae
MR. Effect of sesame oil on the inhibition of
experimental autoimmune encephalomyelitis in C57BL/6
mice. Pakistan journal of biological sciences: Pak J Biol
Sci 2007; 10(11):1790-6.
23. Helft J, Ginhoux F, Bogunovic M, Merad M. Origin and
functional heterogeneity of non-lymphoid tissue dendritic
cells in mice. Immunol rev 2010; 234(1):55-75.
24. Bros M, Jahrling F, Renzing A, Wiechmann N, Dang NA,
Sutter A, et al. A newly established murine immature
dendritic cell line can be differentiated into a mature
state, but exerts tolerogenic function upon maturation in
the presence of glucocorticoid. Blood 2007; 109(9):3820-
G. Mosayebi and SM. Moazzeni
170/ IRANIAN JOURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY Vol. 10, No. 3, September 2011
25. Lu L, Thomson AW. Manipulation of dendritic cells for
tolerance induction in transplantation and autoimmune
disease. Transplantation 2002; 73(1 Suppl):S19-22.
26. Karimi MH, Ebadi P, Pourfathollah AA, Moazzeni SM.
Tolerance Induction by CD40 Blocking through Specific
Antibody in Dendritic Cells. Iran J Allergy Asthma
Immunol 2010; 9(3):141-147.
27. Abediankenari S, Ghasemi M. Generation of Immune
Inhibitory Dendritic Cells and CD4+T Regulatory Cells
Inducing by TGF-b. Iran J Allergy Asthma Immunol
2009; 8(1): 25-30.
28. Steinman RM. The control of immunity and tolerance by
dendritic cell. Pathol boil(Paris) 2003; 51(2):59-60.
29. Li M, Zhang X, Zheng X, Lian D, Zhang ZX, Ge W, et
al. Immune modulation and tolerance induction by RelBsilenced
dendritic cells through RNA interference. J
Immunol 2007; 178(9):5480-7.
30. Xia S, Guo Z, Xu X, Yi H, Wang Q, Cao X. Hepatic
microenvironment programs hematopoietic progenitor
differentiation into regulatory dendritic cells, maintaining
liver tolerance. Blood 2008; 112(8):3175-85.
31. Tiegs G, Lohse AW. Immune tolerance: what is unique
about the liver. J Autoimmun 2010; 34(1):1-6.
32. Du S, Sandoval F, Trinh P, Umeda E, Voskuhl R.
Estrogen receptor-beta ligand treatment modulates
dendritic cells in the target organ during autoimmune
demyelinating disease. Eur J Immunol 2011; 41(1):140-
33. Aghdami N, Gharibdoost F, Moazzeni SM. Experimental
autoimmune encephalomyelitis (EAE) induced by antigen
pulsed dendritic cells in the C57BL/6 mouse: influence of
injection route. Experimental animals / Japanese
Association for Laboratory Exp Anim 2008; 57(1):45-55.
34. Weir CR, Nicolson K, Backstrom BT. Experimental
autoimmune encephalomyelitis induction in naive mice
by dendritic cells presenting a self-peptide. Immunol cell
biol 2002; 80(1):14-20.
35. Huang YM, Yang JS, Xu LY, Link H, Xiao BG.
Autoantigen-pulsed dendritic cells induce tolerance to
experimental allergic encephalomyelitis (EAE) in Lewis
rats. Clin Exp Immunol 2000; 122(3):437-44.
36. Ko HJ, Chung JY, Nasa Z, Chan J, Siatskas C, Toh BH,
et al. Targeting MOG expression to dendritic cells delays
onset of experimental autoimmune disease.
Autoimmunity 2010; 44(3):177-87.
37. Salomon B, Cohen JL, Masurier C, Klatzmann D. Three
populations of mouse lymph node dendritic cells with
different origins and dynamics. J Immunol 1998;
38. Maldonado-Lopez R, De Smedt T, Pajak B, Heirman C,
Thielemans K, Leo O, et al. Role of CD8alpha+ and
CD8alpha- dendritic cells in the induction of primary
immune responses in vivo. J Leukoc Biol 1999;
39. Wu L, Li CL, Shortman K. Thymic dendritic cell
precursors: relationship to the T lymphocyte lineage and
phenotype of the dendritic cell progeny. J Exp Med 1996;
40. Lian ZX, Okada T, He XS, Kita H, Liu YJ, Ansari AA, et
al. Heterogeneity of dendritic cells in the mouse liver:
identification and characterization of four distinct
populations. J Immunol 2003; 170(5):2323-30.
41. O'Connell PJ, Son YI, Giermasz A, Wang Z, Logar AJ,
Thomson AW, et al. Type-1 polarized nature of mouse
liver CD8alpha- and CD8alpha+ dendritic cells: tissuedependent
differences offset CD8alpha-related dendritic
cell heterogeneity. Eur J Immunol 2003; 33(7):2007-13.
42. Bertolino P. Impaired function of dendritic cells
translocating the liver sinusoids: a veto effect
contributing to intrahepatic tolerance? Eur J Immunol
43. Link H, Huang YM, Masterman T, Xiao BG. Vaccination
with autologous dendritic cells: from experimental
autoimmune encephalomyelitis to multiple sclerosis. J
Neuroimmunol 2001; 114(1-2):1-7.
44. Dittel BN, Visintin I, Merchant RM, Janeway CA Jr.
Presentation of the self antigen myelin basic protein by
dendritic cells leads to experimental autoimmune
encephalomyelitis. J Immunol 1999; 163(1):32-9.
45. Ludewig B, Odermatt B, Landmann S, Hengartner H,
Zinkernagel RM. Dendritic cells induce autoimmune
diabetes and maintain disease via de novo formation of
local lymphoid tissue. J Exp Med 1998; 188(8):1493-501.|