| Principal Investigator :
Vineeta Bal Project Associates Ph D Students Collaborators Analysis
of effects of pharmacological agents on the development and activation of
lymphocytes is the theme of research. The aim of this project is to (i) study
the role of modulators of intracellular cAMP levels in T lymphocyte responses
and (ii) analyse the effects of modulators of intracellular cAMP levels on
lymphocyte development. 1.
To study the role of modulators of intracellular cAMP levels in T
lymphocyte responses Using
an allo-recognition system in vitro we had earlier reported that
presence of pentoxifylline (PF), a pan-phosphodiesterase (PDE) inhibitor
during human T cell allo-priming in vitro results in enhanced secondary
response. Presence of PDE inhibitor can result in an increase in intracellular
cAMP levels and hence we analysed effects of various pharmacological agents,
which can modulate cAMP levels in T cells on secondary allo-responses. Since
intracellular cAMP is generated by adenylate cyclase (AC), we used an AC
activator, Forskolin (Fs), as well as an AC inhibitor, SQ22536 (Sq), to
examine the effects of endogenously synthesized cAMP on T cell priming for
secondary responsiveness. When responder T cells were allo-primed by HLA-mismatched
stimulator PBMCs (peripheral blood mononuclear cells) in the presence of 10 µM
Fs for 5 days, harvested and washed before restimulation by irradiated fresh
PBMCs, there was an enhancement in the commitment to secondary responsiveness
similar to that induced by 360 µM PF (Figure-1A). In contrast, the presence
of 100 µM Sq during priming had no effect on T cell commitment to secondary
reactivity (Figure-1B). Further, the presence of the AC inhibitor led to
blockade of the PF-mediated enhancement of secondary responsiveness,
indicating that PF mediates its function through newly synthesised cAMP.
Figure 1: Adenylate cyclase mediated increase in cAMP levels during priming enhances secondary T cell response. T cells primed in presence of Pentoxifylline [A & B] and Forskolin [A] show enhanced secondary response whereas those primed in presence of SQ22536 do not [B]. However, enhancement brought about by pentoxifylline is reversed in presence of SQ22536 [B]. Three
isoforms of PDE, PDE3, 4 and 7 are reported to be expressed in lymphocytes.
Hence, we next analysed the effects of isoform specific inhibitors of PDE.
While PDE4 inhibition by 30 mM Rolipram (Rm) during
allo-priming increased the resultant commitment to secondary response, PDE3
inhibition by 10 mM Trequinsin (Tq) or PDE7 inhibition
by specific anti-sense oligonucleotides showed no such enhancement of T cell
priming. These effects of PF, Rm or Fs were mediated by T cells and not on
APCs as irradiated lightly fixed APCs when used for priming brought about
enhancement in the secondary T cell response. Since
the signalling functions of cAMP are mostly mediated by protein kinase A (PKA),
we next analysed the effects of PKA agonism and antagonism in this system. The
presence of the PKA antagonist RpBrcAMPs (30 µM) by itself during allo
priming did not alter the efficiency of T cell commitment to secondary
reactivity. However, the presence of RpBrcAMPs along with PF (360 µM) during
priming reproducibly blocked the PF-mediated enhancement of secondary response
capability, indicating that the effect of PF on T cell priming was mediated
through cAMP-dependent PKA. We
next asked which of the two major isoform groups of PKA were involved in this
effect of PF on T cell memory commitment. Neither the PKA-I-specific
antagonist RpClcAMPs nor the PKA-II-specific antagonist RpCPTcAMPs had any
effect on T cell priming on their own. However, PKA-I inhibition blocked the
PF-mediated enhancement of T cell priming, while PKA-II inhibition did not.
The lack of a role for PKA-II in the T cell memory-enhancing effect of PF was
also confirmed using an inhibitor of its recruitment to A-kinase anchoring
proteins, AKAP. Together, these data indicated that the enhancement of T cell
commitment to secondary responses brought about by PF was specifically
mediated through PKA-I. Kinases
of the MAPK cascade, activated by the MAPK/extracellular signal-regulated
kinase (ERK) kinase (MEK), are major signalling mediators downstream of cAMP/PKA
in many cell types, although the precise effect of cAMP and PKA on MAPK/ERK
activation may differ. We therefore examined if MEK-1 modulation could modify
the PF-mediated enhancement of T cell priming, using a MEK-1-inhibitor,
PD98059. The presence of PD98059, either alone or along with PF, during T cell
allo priming did not modify the subsequent secondary T cell responsiveness. While
PF functions via cAMP and PKA to enhance T cell priming, cAMP-PKA-mediated
signals do not normally appear to be regulating T cell memory commitment,
since inhibition of AC or PKA during T cell priming does not affect secondary
responsiveness in the absence of PF. Since it has been shown that the
cAMP-PKA-mediated signalling pathway is triggered early during T cell
activation, it was possible that the PF-cAMP-PKA-mediated effect on T cell
priming was due to modulation of late events in T cell activation. We tested
this possibility by adding PF to T cell priming cultures during restricted
time periods. During the 96-h period of T cell priming in vitro,
presence of PF during the first 48 h had no effect on the magnitude of recall
responses, whereas addition of PF late during priming over the 48-96 h period
showed enhancement of priming equivalent to that seen if PF was present all
through the priming period. Similarly, the presence of Rm and dbcAMP during
this 48-96 h window also enhanced T cell priming. Thus, PF-cAMP-PKA signalling
may either extend an early event or modulate a late event in T cell priming to
enhance secondary responsiveness. We
have shown previously that death of allo-primed T cells in culture is
inhibited by PF. It has been reported that activated primary T cells are not
susceptible to apoptotic cell death at early time points. It was therefore
possible that the late event in T cell activation affected by PF to enhance T
cell memory involved apoptosis. We tested the effect of PF on T cell apoptosis
directly by triggering activation-induced cell death (AICD) in T cells with
anti-CD3 mAb, and examining the effect of PF on this event. At 48 h
post-stimulation, anti-CD3-triggered T cells showed induction of caspases
(Figure-2A), the cysteine aspartyl proteases thought to play a central role in
many pathways of apoptosis, as well as membrane changes characteristic of
apoptosis as indicated by binding of annexin-V (Figure-2B). The presence of PF
in culture inhibited the induction of both caspases and apoptosis, suggesting
that apoptosis is likely to be the event affected by PF during T cell
activation for enhancing secondary response commitment.
Figure 2
:
Presence of pentoxifylline prevents induction of caspases [A]
and death as measured by annexin V staining [B] following anti-CD3 activation.
Shaded curve -control, thin line - anti-CD3 activation without PF, thick line
- anti-CD3 activation in presence of PF. 2
To analyse the effects of modulators of intracellular cAMP levels on
lymphocyte development We
have reported earlier that presence of PF or dibutyryl (db)cAMP during
reconstitution of the thymus following sublethal irradiation results in a
block in the development from double negative (CD4-8-) to double positive
(CD4+8+) stage. There are many similarities in the development of T and B
lineages including use of recombination activating genes (RAG). Hence we
attempted to look at the bone marrow reconstitution and development of B
lineage cells following sublethal irradiation in presence of PF. Mice were
irradiated at 400 rads and bone marrow was harvested and stained for B lineage
cells with antibody to B220 and for surface or intracytoplamic m-heavy
chain for identifying various stages of B cell development. Until day 7 post
irradiation mature B cells (B220+, IgMhi) are not developed though pre-B cells
(B220+, surface IgM-) are seen in comparable proportions in mice receiving
saline or PF (8.2% + 1.2 vs. 14.03 + 1.63). The proportion of
B220+ cells expressing m-chain either on the surface or
intracellularly (i.e. to include pro- and pre-B cells) is also comparable in
the two groups though the number of B220+ cells in these two groups are much
lower than in unirradiated mice. By day 21 irradiated mice receiving saline
show comparable numbers of mature B cells to normal unirradiated mice.
However, irradiated mice receiving PF show a smaller percentage of pre-B cells
and pro-B cells as compared to saline treated mice. These preliminary findings
indicate that reconstitution of the bone marrow B cells might be affected in
presence of PF similar to the effects seen on thymocyte development. Publications Original
peer-reviewed articles 1.
Mukherjee P, Dani A, Bhatia S, Singh N, Rudensky AY, George A, Bal V,
Mayor S and Rath S (2001) Efficient presentation of both cytosolic and
endogenous transmembrane protein antigens on MHC class II is dependent on
cytosoplasmic proteolysis. J Immunol 167:2632-2641. 2.
Bhatia S, Mukhopadhyay S, Jarman E, Hall G, George A, Basu SK, Rath S,
Lamb JR and Bal V (2002) Scavenger receptor-specific allergen delivery elicits
IFN-g-dominated
immunity and directs established TH2-dominated
responses to a non-allergic phenotype. J Allergy Clin Immunol 109:321-328. 3.
Mukhopadhyay S, Mohanty M, Mangla A, George A, Bal V, Rath S and
Ravindran B (2002) Macrophage effector functions controlled by Bruton’s
tyrosine kinase are more crucial for microfilarial clearance than the cytokine
balance of T cell responses. J Immunol 168:2914-2921. |
