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The cytokine release which follows the interaction of recipient dendritic
cells (DC) and donor lymphocytes (TL) initiates the immune processes which
culminate in graft rejection. Cell-cell contact between DC and TL results from
a number of surface proteins, including CD86 and CD80 on DC and the receptor
CD28 on TL. While CD28 co-stimulatory signals can be mediated via tyrosine
phosphorylation signaling, the nature of the DC response following TL contact
remains to be defined. We postulated that such contact leads to DC tyrosine
phosphorylation and therefore to activation of key signaling cascades, such as
the Mitogen Activated Protein (MAP) kinases. To test this hypothesis splenic TL
harvested from C3H mice were layered over confluent DC isolated from C57BL/6
mice and co-cultured. TL and DC were separated at various times, and the two
cell populations studied individually for signaling events. As determined by
Western blot analysis, the TL stimulated a marked increase in tyrosine
phosphorylation within DC that peaked by 90 minutes and then faded. By
contrast, tyrosine phosphorylation in the TL population over the same time span
was weak. Phosphorylated, active ERK MAP kinase isoforms were consistently
upregulated in DC, with less phosphorylation of the related p38 MAP kinase. The
physiologic importance of DC tyrosine phosphorylation was suggested by a
67±11% (mean±SEM, n=4) inhibition of 24-hour TNF
and 18±9%
inhibition of IL-2 production when the TL / DC co-culture was exposed to the
broad-spectrum tyrosine kinase inhibitor Tyrphostin AG126 (50µM).
Intriguingly, selective ERK MAP kinase inhibition with PD98059 (50µM)
inhibited production of both TNF_ (45±10%) and IL-2 (39±6%), while
selective p38 inhibition with SB203580 (30µM) inhibited only TNF (52±8%), with no
effect on IL-2 release (<1%). We have previously noted that blockade of the
CD86/ CD28 and CD80/CD28 interaction with specific monoclonal antibodies
against CD86 and CD80 abrogates cytokine generation in this model. Pretreatment
of DC with the same antibodies markedly attenuated TL -induced DC tyrosine
phosphorylation. These data argue that contact with allogeneic TL stimulates a
time-dependent increase in DC tyrosine phosphorylation that is mediated at
least in part via CD86 and CD80 and is crucial for the subsequent induction of
immune responses. The ERK MAP Kinase in particular plays a key role in the
allogeneic immune response, suggesting that future immunomodulatory therapies
could be targeted to this pathway.