Objective. Rituximab is a therapeutic anti-CD20 antibody used for in vivo depletion of B cells in proliferative and autoimmune diseases. However, the mechanisms of action are not fully understood, since not all of the therapy-mediated effects can be explained by the depletion of antibody-secreting cells. In addition to B cells, there is also a small population of T cells coexpressing CD20 in all individuals. This study was conducted to examine the phenotype and function of CD3؉CD20؉ T cells in patients with rheumatoid arthritis (RA) and healthy controls.Methods. The phenotype and apoptosis of peripheral blood mononuclear cells from healthy donors and RA patients were examined by 4-color fluorescenceactivated cell sorting analyses. Cytokine production was determined by intracellular staining and measurement of cytokines in the supernatants. Proliferation of sorted T cell populations was analyzed using 3 H-thymidine uptake assays.Results. In healthy individuals, 0.1-6.8% of peripheral blood T cells (mean 1.6%; n ‫؍‬ 142) coexpressed CD20, which was not significantly different from that in the peripheral blood of RA patients, in whom 0.4-2.6% of T cells (mean 1.2%; n ‫؍‬ 27) were CD20؉. During rituximab therapy, the CD20؉ T cells along with the B cells were eliminated from the RA peripheral blood. Among the CD20؉ T cells, 45% coexpressed CD8 and 55% coexpressed CD4. Polyclonal CD3؉CD20؉ cells were functionally characterized by constitutive cytokine production (i.e., interleukin-1␤ and tumor necrosis factor ␣), a low proliferative capacity, a high activation state, and enhanced susceptibility to apoptosis.Conclusion. These findings suggest that CD20؉ T cells represent a terminally differentiated cell type with immune-regulatory and proinflammatory capacities. Depletion of CD20؉ T cells may be an additional mechanism by which anti-CD20 therapy functions in patients with RA.

An anti-tumor-promoting effect of indomethacin and related nonsteroidal anti-inflammatory drugs (NSAIDs) as well as the ability of the tumor promoter 12-O-tetradecanoylphorbol-13- acetate (TPA) to increase the level of prostaglandins in murine keratinocytes and mouse epidermis in vivo has been repeatedly documented. Here, the expression of prostaglandin H synthase (PGHS) isozymes, which are major targets of NSAIDs, was investigated in different stages of tumor development in mouse skin. Mouse epidermis in vivo constitutively expressed PGHS-1. PGHS-1 steady-state levels remained unchanged upon induction of acute or chronic epidermal hyperplasia by TPA and in papillomas and carcinomas generated by the initiation-promotion procedure, with 7,12-dimethylbenz[a]anthracene as initiator and TPA as promoter. Thus, the elevated prostaglandin level in the acute hyperplastic epidermis was very likely due to PGHS-2 induction. Repeated applications of TPA resulted in stationary hyperplasia and downregulation of PGHS-2 expression and prostaglandin levels, suggesting that the epidermis had adapted to the TPA stimulus. In papillomas and carcinomas, however, constitutive overexpression of PGHS-2 was found, with a large amount of prostaglandin E2 and prostaglandin F2 alpha. Keratinocyte cell lines corresponding to different stages of tumor development also constitutively over-expressed PGHS-2. Considered with inhibitor studies, these data suggest that PGHS-2 has a critical role in skin carcinogenesis. The anti-tumor-promoting effect of the PGHS inhibitor indomethacin is specifically reversed by prostaglandin F2 alpha, indicating that this prostaglandin type has a significant role in tumor development.

Normal mouse epidermis constitutively expresses prostaglandin-H synthase 1 (PGHS-1) but no PGHS-2. Acute inflammation and epidermal hyperplasia, (hyperplastic transformation), as evoked in adult mouse skin in vivo by wounding or by the phorbol ester phorbol 12-myristate 13-acetate (PMA), resulted in a transient induction of PGHS-2 expression while PGHS-1 remained unchanged. Under conditions of a stationary epidermal hyperplasia, as in neonatal mouse epidermis, PGHS-1, but not PGHS-2, expression was observed. Induction of 'balanced hyperproliferation' by 4-O-methyl-phorbol 12-myristate 13-acetate (4-O-methyl-PMA) did not lead to PGHS-2 expression. When keratinocytes were isolated from neonatal mouse skin and separated by Percoll density-gradient centrifugation according to their stage of differentiation, PGHS-1 mRNA expression and protein were found to be highest in the differentiated cells compared with those from the proliferative compartment. A similar distribution of PGHS-1 mRNA was found in keratinocytes from adult mice, whereas PGHS-1 protein was equally distributed in all cell types. Contrary to the situation in intact epidermis, PGHS-2 mRNA but no protein was detected in all cell fractions. Established keratinocyte lines constitutively expressed both isoenzymes at different ratios. In the mouse line MSCP5 an almost exclusive expression of PGHS-2 was found, which was further enhanced by PMA treatment. These data indicate that the expression of PGHS-2 in mouse epidermis is specifically related to the emergency reaction of hyperplastic transformation.