The expression of peripheral benzodiazepine receptors in human skin: the relationship with epidermal cell differentiation.

The peripheral benzodiazepine receptor (PBR) is a protein of mitochondrial outer membranes utilizing porphyrins as endogenous ligands. PBR is part of a heteromeric receptor complex involved in the formation of mitochondrial permeability transition pores and in the early events of apoptosis. PBR may function as an oxygen-dependent signal generator; recent data indicate that these receptors may preserve the mitochondria of haematopoietic cell lines from damage caused by oxygen radicals. To identify PBRs in human skin, we used a specific monoclonal antibody directed against the C-terminus fragment of the human receptor. PBR immunoreactivity was found in keratinocytes, Langerhans cells, hair follicles and dermal vascular endothelial cells. Interestingly, confocal microscopic examination of skin sections revealed that PBR expression was strongly upregulated in the superficial differentiated layers of the epidermis. Ultrastructurally, PBRs were distributed throughout the cytoplasm but were selectively expressed on the mitochondrial membranes of epidermal cells. The elevated level of PBRs in the spinous layer was not associated with an increased number of mitochondria nor with an increased amount of mRNA as assessed by in situ hybridization on microautoradiographed skin sections. The present work provides, for the first time, evidence of PBR immunoreactivity in human skin. This mitochondrial receptor may modulate apoptosis in the epidermis; its increased expression in differentiated epidermal layers may represent a novel mechanism of natural skin protection against free radical damage generated by ultraviolet exposure.

Quantitative method to determine mRNA levels by reverse transcriptase-polymerase chain reaction from leukocyte subsets purified by fluorescence-activated cell sorting: application to peripheral cannabinoid receptors.

BACKGROUND: While cytometry is widely used in the detection of cell proteins, its application to quantitative evaluation remains problematic when target proteins or receptors are weakly expressed in cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) is a technique whose sensitivity and specificity make it appropriate for analyzing nucleic acids and thus genes expressed in cells. Combining these two techniques, we developed a method to quantify the transcript expression of the peripheral cannabinoid receptor (CB2-r) in peripheral blood lymphocyte subpopulations and in tonsillar B-cell subpopulations. METHODS: This strategy first involves quantitative RT-PCR performed kinetically, followed by enzyme detection of PCR products using an oligonucleotide probe sandwich-hybridization assay onto microplates. RESULTS: B cells exhibit CB2-receptor mRNA levels 10 times higher than those of other lymphocyte subsets. Using this technique, we observed a modulation of CB2-r mRNA level following tonsillar B-cell differentiation. Lastly, this new technology was validated by comparing the mRNA levels of CB2-r with the expression of CB2-r proteins assayed by flow cytometry, using specific CB2-r antibody labelling. CONCLUSIONS: This method allows precise measurement of the mRNA of CB2-r performed on cell numbers as low as 10(5) after sorting. Its performance, high accuracy, reproducibility, and reliability make it a valuable tool for assaying proteins weakly expressed in cells.

Modulation and functional involvement of CB2 peripheral cannabinoid receptors during B-cell differentiation.

Two subtypes of G-protein-coupled cannabinoid receptors have been identified to date: the CB1 central receptor subtype, which is mainly expressed in the brain, and the CB2 peripheral receptor subtype, which appears particularly abundant in the immune system. We investigated the expression of CB2 receptors in leukocytes using anti-CB2 receptor immunopurified polyclonal antibodies. We showed that peripheral blood and tonsillar B cells were the leukocyte subsets expressing the highest amount of CB2 receptor proteins. Dual-color confocal microscopy performed on tonsillar tissues showed a marked expression of CB2 receptors in mantle zones of secondary follicles, whereas germinal centers (GC) were weakly stained, suggesting a modulation of this receptor during the differentiation stages from virgin B lymphocytes to memory B cells. Indeed, we showed a clear downregulation of CB2 receptor expression during B-cell differentiation both at transcript and protein levels. The lowest expression was observed in GC proliferating centroblasts. Furthermore, we investigated the effect of the cannabinoid agonist CP55,940 on the CD40-mediated proliferation of both virgin and GC B-cell subsets. We found that CP55,940 enhanced the proliferation of both subsets and that this enhancement was blocked by the CB2 receptor antagonist SR 144528 but not by the CB1 receptor antagonist SR 141716. Finally, we observed that CB2 receptors were dramatically upregulated in both B-cell subsets during the first 24 hours of CD40-mediated activation. These data strongly support an involvement of CB2 receptors during B-cell differentiation.