Effect o f cAMP elevation on DC differentiation
4.3.4 CD40 ligation does not induce D C l differentiation
The literature suggests contrasting results for the role o f CD40 ligation in generating
an IL-12 producing, D C l phenotype (Celia et aL, 1996; Hilkens et aL, 1997; Koch et
aL, 1996; Snijders et aL, 1998). This may reflect differences in sources o f DCs,
culture conditions and/or the extent o f CD40 engagement in these studies. In order to
assess whether CD40 engagement on DCs leads to differentiation into a D C l
phenotype, BmDC were stimulated with either aCD40 alone, or together with LPS
(positive control for D C l inducer). The function o f these DCs in skewing the
subsequent T hl/Th2 responses was assessed by analysing the cytokines produced by
D O ll.lO Tg CD4'*' CD62L^'^^ T cells. Stimulation w ith DCs treated with varying
concentrations o f aCD40 did not change the cytokine profile o f these CD4^ T cells,
although there was a marginal decline in IL-4 production with increasing
concentration o f aCD40 (figure 4.9A and C). Furthermore, aCD40 triggering o f LPS-
stimulated DCs had no effect on the D C l phenotype mediated by LPS (figure 4.9B
and D).
In addition, CD40 ligation o f DCs stimulated with PGE2 or dbcAMP did not result in
an enhancement o f IL-4 secretion from D O ll.lO Tg CD4^ T cells, while there was a
slight enhancement o f IFNy levels (figure 4.10). Thus, it appears that CD40 ligation
does not result in DC1/DC2 differentiation o f DCs in this system, but rather acts as an
IFNy 1000 900 800 g 700 g 600 500 U_ 400 300 200 100 0 IL-4 A o [OVA] = 0 o [OVA] = 100pM . [OVA] = 10nM 0 0,02 0.03 0.06 0.13 0.25 0.5 1 8 16 [aCD40] (ig/ml C 0 0.02 0.03 0.06 0.13 0.25 0.5 1 2 4 [aCD40] |ag/ml 1000 900 800 700 600 500 400 300 200 100 0 _ 600 g 500 B (+ L PS) 0 0.02 0.03 0.06 0.13 0.25 0.5 1 [aCD40] |ag/ml D (+LPS) 8 16 0 0.02 0.03 0,06 0.13 0.25 0.5 1 2 4 8 16 [aCD40] |ag/ml
Figure 4.9 CD40 ligation does not induce D C l differentiation.
Day 5 BmDC (Balb/c) were stimulated overnight with titrated concentrations of anti- CD40 antibody (aCD40) with or without LPS (l)ig/ml). CD62L*’’®'’ CD4+ T cells (1x10^) from DOll.lOTg mouse spleens were stimulated with these DCs (1x10"^) in the presence (grey circles lOOpM, black circles lOnM) or absence (open circles) of OVA323-339 for 7 2
hours. Cells were then stimulated with PMA (50ng/ml) and ionomycin (500ng/ml) for 8
hours before supernatants were removed for cytokine analysis by ELISA. IFNy
production by CD4+ T cells in response to DCs stimulated with aCD40 alone (A), or DCs stimulated with aCD40 and LPS (B), and IL-4 production from CD4+ T cells in response to DCs stimulated with aCD40 alone (C), or DCs stimulated with aCD40 and LPS (D), are shown. These data are means of duplicate wells from one representative experiment out of three performed. SD < 5% in all cases.
IFNy IL-4 2 5 0 0 2000 1 1500 t 1000 - 5 0 0 -
medium aCO40 PGE, PGE; dbcAMP dbcAMP + aCD40 + aCD40
medium aCD40 PGE, PGE; dbcAMP dbcAMP + aCD40 + aCD40
Figure 4.10 CD40 ligation does not enhance production of IFNy or IL-4 by or
[)ÇdbcAMP^
D ay 5 Bm DC (Balb/c) were stimulated overnight with m edium alone, PGE; ( 10"^ M ), or dbcAM P (10"^ M) with or without aC D 40 (2|ig/m l), C D 62L ‘'*8h C134+ j cells (1x10^) from D O ll.lO Tg mouse spleens were stimulated with these DCs (1x10"^) in the presence o f OVA323-339 (lOOnM) for 72 hours. C ells were then stimulated with PM A (50ng/m l) and ionom ycin (500ng/m l) for 8 hours before supernatants w ere rem oved for cytokine analysis by ELISA. These data are means o f six experiments (±SE).
4.4 Discussion
Increasing evidence suggests that DCs can differentiate into distinct phenotypes
which can trigger T hl (D C l) or Th2 (DC2) responses (Kalinski et aL, 1999; Moser
and Murphy, 2000; Reid et aL, 2000). This offers a potentially attractive method o f
targeting appropriate types o f immune responses to combat various types o f diseases.
However, the mechanisms o f this functional differentiation o f DCs are poorly
understood. Based on the hypothesis that DC1/DC2 differentiation occurs upon
recognition o f pathogen products and infection/injury-mediated factors, the effects o f
elevation o f intracellular cAMP and CD40 ligation on DC function were studied in
this chapter.
It was found that an elevation o f intracellular cAMP induced by PGB2, dbcAMP,
forskolin, CTx and PTx did not correlate with DC maturation (figure 4.2 and 4.3) or
T hl/T h2 cytokine patterns produced by responding CD4^ T cells (figure 4.4 and 4.5).
In fact, CTx and PTx were the only cAMP elevating reagents found to induce both
maturation o f DCs, as measured by surface molecule expression and MLRs, and
induction o f T cell cytokine responses. Both DC^^^ and DC^^^ induced increased
IFNy production from responding CD4^ T cells but not lL-4 production, suggesting
that these DCs exhibited a D C l phenotype. CD40 ligation on DCs resulted in
enhanced proliferation (allogeneic MLR, figure 4.7) and lL-2 production (D O ll.lO
Tg, figure 4.8) o f responding T cells, but had no effect on T hl/T h2 differentiation as
measured by cytokine secretion (figure 4.9 and 4.10).
Based on early studies that indicated a role o f cAMP elevating reagents in DC1/DC2
Rieser et al., 1997; Steinbrink et al., 1997), five reagents known to elevate cAMP
were tested in this study: PGE2, dbcAMP, forskolin, CTx and PTx. The molecular
mechanisms regulating the enzyme responsible for cAMP synthesis, adenylate
cyclase, involve a complex coupling system with different stimulatory or inhibitory
receptors and corresponding GTP binding proteins, or G-proteins (Gs and GJ. A G-
protein is composed of three different polypeptide chains, a , (3, and y. The a chain o f
Gs proteins binds GTP and activates adenylate cyclase, while binding o f the a chain
o f Gi proteins inhibits the activation o f this enzyme. This process is tightly regulated
by hydrolysis o f GTP to GDP, which inactivates the ability o f G-proteins to bind to
adenylate cyclase. This activation process o f adenylate cyclase is schematically
illustrated in figure 4.11.
Plasm a m em brane G-protein complex
ATP
cAMP
Receptor protein Adenylate cyclase
Figure 4.11 Process of adenylate cyclase activation.
Ligand (e.g., PGE2) binding alters the conformation of receptors ((D), causing the receptor to bind
to a G protein (@). This reduces the affinity of G protein for GDP. GDP then dissociates, allowing GTP to bind (0 ). This causes Ga to dissociate from the G protein complex, allowing binding to adenylate cyclase (®). This leads to activation of the enzyme and cAMP synthesis results (0 ). Meanwhile, dissociation of the ligand returns the receptor to its original conformation (®). GTP is hydrolysed (@) and causes the G protein to dissociate from the adenylate cyclase (which becomes inactive) (®).
While forskolin acts on adenylate cyclase directly to activate the enzyme, CTx, PTx
and PGE2 act upon the complex o f molecules that activate the enzyme (dbcAMP is an
cAMP analogue). Cholera toxin mediates ADP ribosylation o f as, which prevents
hydrolysis o f GTP, resulting in continuous activation o f adenylate cyclase. In
contrast, pertussis toxin catalyses the ADP ribosylation o f aj. This prevents the G,
complex from interacting with receptors, and as a result the complex remains bound
to GDP and is unable to inhibit adenylate cyclase activity. PGE2 interacts with
specific membrane receptors coupled to Gs protein, which in turn activates adenylate
cyclase.
The effects o f a range o f cAMP inducers and other modulins studied in this thesis on
DC function are summarised in table 4.1.
Table 4.1 Changes in function of DCs upon differential stimulation (relative to DCO).
DCPGEz QQdbcAMP QQforskolin DC™ DCPTx DCLps* DCES62** DC maturation markers ++ +/— + / - +++ +++ ++++ +++ Allogeneic MLR - - ++ +++ +++ ++
Syngeneic MLR - - - ++++ - -
IL-12 production
DO11,10 Tg C D 4 + 1 cell cytokine responses
- + /- + ++ +/—
IL-2 - - - ++ ++ ++
IFNy + /- + / - ++ ++++ +++ -
IL-4 - - - +++
* DCLPs is Included as stimulator o f DC1, while ** is typical of DC2 phenotype [(Whelan et al., 2000); data not shown]. Scale: - denotes no difference from DCO phenotype; ++++ denotes maximum expression/response obtained within the systems tested; each + accounts for % of the maximum expression/response observed.
The data shows clearly that whilst CTx and PTx induced both maturation and
differentiation o f DCs, other cAMP inducers (PGE2, forskolin and dbcAMP) did not.
Thus, this suggests that an increase in intracellular cAMP level is not likely to be
responsible for the resulting maturation o f DCs in response to CTx and PTx, as not all
cAMP inducers caused maturation o f DCs. This also implies that a component o f
CTx and PTx other than that induces cAMP elevation is responsible for maturation
stimulus o f DCs, necessary to achieve functional differentiation o f DCs. In contrast
to the work presented in this chapter, earlier studies implicated the role o f elevating
cAMP in DCs differentiation by examining the effects o f cAMP inducers on human
monocyte-derived DCs (Gagliardi et al., 2000; Kalinski et al., 1997a; Kalinski et al.,
1997b; Rieser et al., 1997; Steinbrink et al., 1997). Thus the differences in the type o f
DCs may explain the different results obtained between this study and earlier studies.
The importance o f the interaction between CD40L on activated T cells and CD40
expressed on B cells, monocytes and DCs in enhancement o f APC function has been
well documented (van Kooten and Banchereau, 1997). CD40 ligation on BmDC
resulted in a small, but significant enhancement o f the allogeneic M LR (figure 4.7)
and lL-2 production from D O ll.lO Tg CD4^ T cells (figure 4.8). However, when
aCD40 was used in conjunction with LPS to stimulate DCs (DcLPS/aCD40^^ both o f
these responses were amplified in an additive manner. Interestingly, the response
correlated with the level o f costimulatory molecules expressed on DCs (Chapter 3,
figure 3.5). failed to induce T hl or Th2 responses, as indicated by the levels
o f IFNy and fL-4 produced by responding CD4^ T cells (figure 4.9). Furthermore, in
contrast to the amplifying role on allogeneic M LR and lL-2 production induced by
not observed, suggesting that ligation o f CD40 on DCs has an insignificant influence
on T hl/T h2 differentiation (figure 4.9). Similarly, enhancement o f T hl/Th2
responses by and was not observed upon ligating CD40 on these
DCs (figure 4.10).
An enhanced production o f IL-12p70 by was not observed in this study
(figure 4.6). This agrees with earlier studies that demonstrated that mere ligation o f
CD40 on DCs is not sufficient to trigger production o f this cytokine (Hilkens et al.,
1997; Snijders et a l, 1998). This is not surprising, given that CD40L is expressed on
both T hl and Th2 cell types. It would be potentially dangerous if, as a result o f any
DC-T cell interactions, DCs only acquired an ability to produce high levels o f IL-12
and induced T hl responses. Thus, T hl responses may require strict regulation, and
subsequently IL-12 production by DCs may be under stringent control. The
differences in the literature relating to the role o f CD40 ligation on IL-12 production
by DCs may reflect the differences in methods used for preparation o f DCs, lineages
o f DCs, methods o f CD40 ligation used and/or time period at which the cytokine was
measured.
The two studies that agree with the observations presented in this chapter examined
the response o f human monocyte-derived DCs to soluble CD40L. In contrast, studies
indicating a direct role o f CD40 ligation in the induction o f IL-12 used a CD40L-
transfected cell line to stimulate human monocyte-derived DCs (Celia et al., 1996), or
an antibody against CD40 (aCD40) to stimulate mouse splenic DCs (Koch et al.,
1996). In addition, it is likely that the extent o f DC maturation in these two studies
shown to take a longer period than human DCs for maturation in response to aCD40
(Kelleher and Beverley, 2001; Koch et al., 1996). Thus, it is possible that DCs
stimulated with aCD40 in the work presented in this chapter may have an enhanced
secretion o f IL-12 at a later time point. In addition, since low levels o f contamination
with endotoxin may be sufficient to trigger maturation o f DCs, high levels o f IL-12
production in response to ligation o f CD40 in some o f the previous studies may be
due to the presence o f endotoxin.
One o f the most surprising findings in this chapter is that DC^^^ triggered a potent
syngeneic MLR at a level almost comparable to that o f the allogeneic MLR. The
generation o f such a syngeneic response has not been described before by DCs
stimulated with other modulins tested. Both PTx and CTx have been used as mucosal
adjuvants in vaccine trials (Hormozi et a l, 1999; Lycke, 1997; Rappuoli et a l, 1999).
However, the mechanism o f their adjuvanticity remains largely unknown. It is
plausible that these toxins activate the immune system by activating DCs. This is
further investigated in the following chapter in this thesis.
In summary, the D C l phenotype resulted from stimulation with bacterial toxins
including LPS, CTx and PTx, all o f which induced maturation o f DCs. In contrast,
other cAMP inducers that failed to activate DCs had no effect on DC1/DC2
differentiation. In addition, CD40 ligation resulted in amplification o f some o f T cell
responses (proliferation and IL-2 production), but not differential cytokine
production. As CD40 does not function as a pattern recognition receptor (PRR), these
results are consistent with the hypothesis that modulins that are recognised by the
chapter is that, at least for murine BmDC, a maturation-driving signal is a prerequisite
o f the modulins for functional differentiation o f DCs. It is likely that these signals are