Biology Reference
In-Depth Information
PKA pathway, and this was observed in a variety of early studies (Kammer
1988
).
An analysis of isolated T cells comparing PDE inhibitor sensitivity of secretory
functions and blastogenesis with PDE isoenzyme profiles was first performed by
Robiscek showing that both PDE3 and PDE4 are present in T cells (Robicsek et al.
1989
,
1991
). T-cell isolation in those days was a complicated, time-consuming
multi-step procedure with low yield and the risk for activating quiescent cells.
Later, a standardised technique for cell isolation and measurement of intracellular
PDE activities was developed (Gantner et al.
1997b
, Schudt and Tenor 1996),
which by using selective inhibitors, allowed an approach for determining PDE
isoenzyme profiles in isolated CD4+ and CD8+ T cells or macrophages (Tenor et al.
1995b
,
c
). Functionally, it was again demonstrated that cytokine release was partly
inhibited by either PDE3 or PDE4 inhibitors, whereas complete inhibition was
observed in the combined presence of both inhibitors or dual-selective PDE3/4
inhibitors such as zardaverine. This indicated that PDE3 and PDE4 participate in
regulation of the cAMP pathway in T cells.
Alveolar macrophages showed similar inhibitory patterns (Schade and Schudt
1993
; Tenor et al.
1995c
), but since they were difficult to obtain they were
differentiated in cell culture together with dendritic cells from blood MC (Gantner
et al.
1997a
). In parallel to phenotypic differentiation, PDE1 and PDE3 activities
were augmented whereas PDE4 activity - the major activity in MC - declined, and
MC-derived macrophages acquired the identical PDE activity profile as found for
alveolar macrophages. Functionally, simultaneous inhibition of PDE3 and PDE4
was most effective in inhibition of TNF
a
release from both MC-derived cell
populations (Gantner et al.
1999
).
4.4 Does Exposure of Inflammatory Cells to ß-Mimetics
Evoke Hyperreactivity?
Absolute PDE activities in cells and tissues were of considerable interest; however,
all these determinations of cellular enzyme activities were based on defined sub-
strate concentrations and enzymological standard conditions. PDE profiles as
described in many papers were useful for comparing different tissues and for
explaining the functional effects of mono- or dual-selective PDE inhibitors. On
the contrary, it was obvious that PDE activities in situ were regulated at various
levels: (1) substrate concentrations and kinetic behaviour, (2) concentration of
competitors and endogenous inhibitors, (3) regulation by phosphorylation and/or
other modifications, (4) intracellular compartmentalisation and membrane associa-
tion and (5) degradation and de novo synthesis. An upregulation of PDE4 could be a
sufficient explanation for an enhanced activity state of PDE4-containing cells.
Indeed, such enhanced PDE4 activity levels in MC and/or lymphocytes from
atopic patients were described by Hanifin in several papers (Holden et al.
1986
).
Unfortunately, these claims were not reproducible by others (Gantner et al.
1997a
,
b
).
