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Updated: Jul 18, 2026
Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture
Published on: April 8, 2016
Immunochemical characterization of the distinct monocyte cyclic AMP-phosphodiesterase from patients with atopic
S C Chan1, D Reifsnyder, J A Beavo
1Department of Dermatology, Oregon Health Sciences University, Portland 97201.
Background:
Previous findings have suggested that the immunopathology of patients with atopic dermatitis (AD) results from altered cellular responses caused by cyclic nucleotide regulatory abnormalities. One such defect is the increased degradation of the second messenger, cyclic adenosine monophosphate (cAMP), by elevated cAMP-phosphodiesterase (PDE) activity in patients with AD.
Methods:
We used two monoclonal antibodies to identify the major PDE isoform in AD blood monocytes. We have also characterized the abnormal PDE activity by means of chromatofocusing and sucrose gradient centrifugation.
Results:
The chromatofocusing technique allowed the separation of a PDE-containing fraction (isoelectric point = 6.1) from AD monocytes but not from normal cells. This monocyte fraction accounted for most of the elevated leukocyte-PDE activity and was a cytosolic, cAMP-specific, low Michaelis constant, calcium-calmodulin-dependent enzyme, inhibited by the cAMP-PDE inhibitor, Ro 20-1724. The majority of the PDE activity in this chromatofocused fraction was immunoadsorbed by the solid-phase immobilized antibodies against calcium-calmodulin-dependent PDE.
Conclusions:
The increased degradation of cAMP by a unique form of PDE may cause defective regulation of intracellular functions of AD monocytes, leading to the characteristic hyperreactive immune and inflammatory events. Characterization of PDE isoenzymes from different leukocyte subpopulations may allow further expansion of cell-directed therapy for inflammatory disease.
Insights
Patients with atopic dermatitis (AD) have elevated cyclic adenosine monophosphate-phosphodiesterase (cAMP-PDE) activity in monocytes. This unique PDE isoform contributes to immune dysregulation in AD, offering a target for new therapies.
Area of Science:
- Immunology
- Biochemistry
- Dermatology
Background:
- Atopic dermatitis (AD) immunopathology linked to cyclic nucleotide regulatory defects.
- Elevated cyclic adenosine monophosphate-phosphodiesterase (cAMP-PDE) activity increases cAMP degradation in AD patients.
Purpose of the Study:
- Identify the primary PDE isoform in AD blood monocytes.
- Characterize the abnormal PDE activity in AD monocytes.
Main Methods:
- Utilized monoclonal antibodies for PDE isoform identification.
- Employed chromatofocusing and sucrose gradient centrifugation to characterize PDE activity.
Main Results:
- A unique PDE fraction (isoelectric point = 6.1) was isolated from AD monocytes, absent in normal cells.
- This fraction represented the majority of elevated leukocyte-PDE activity and was identified as a cytosolic, cAMP-specific, calcium-calmodulin-dependent enzyme.
- The enzyme was inhibited by Ro 20-1724 and immunoadsorbed by antibodies against calcium-calmodulin-dependent PDE.
Conclusions:
- Increased cAMP degradation by this unique PDE may impair AD monocyte function, causing hyperreactive immune responses.
- Characterizing PDE isoenzymes in leukocyte subpopulations could advance cell-directed therapies for inflammatory diseases.
